Secreted salivary zsig32 polypeptides

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for secreted salivary zsig32 polypeptides. The polypeptides, and polynucleotides encoding them modulate adhesion or modulate or indicate salivary gland function. The present invention also includes antibodies and binding proteins for the zsig32 polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/040,786, filed Mar. 18, 1998, and claims the benefit ofProvisional U.S. Patent Application No. 60/041,263, filed Mar. 19, 1997.

BACKGROUND OF THE INVENTION

The salivary glands synthesize and secrete a number of proteins havingdiverse biological functions.

Such proteins facilitate lubrication of the oral cavity (e.g., mucinsand proline-rich proteins), remineralization (e.g., statherin and ionicproline-rich proteins) and digestion (e.g., amylase, lipase andproteases) and provide anti-microbial (e.g., proline-rich proteins,lysozyme, histatins and lactoperoxidase) and mucosal integritymaintenance (e.g., mucins) capabilities. In addition, saliva is a richsource of growth factors synthesized by the salivary glands. Forexample, saliva is known to contain epidermal growth factor (EGF), nervegrowth factor (NGF), transforming growth factor-alpha (TGF-α),transforming growth factor-beta (TGF-β), insulin, insulin-like growthfactors I and II (IGF-I and IGF-II) and fibroblast growth factor (FGF).See, for example, Zelles et al., J. Dental. Res. 74(12): 1826-2, 1995.Synthesis of growth factors by the salivary gland is believed to beandrogen-dependent and to be necessary for the health of the oral cavityand gastrointestinal tract.

Some salivary gland-produced proteins, such as EGF, are believed to havesystemic wound healing effects. Effective wound healing appears torequire extended exposure of afflicted tissue to growth factors, whichmay be facilitated in the oral cavity and gastrointestinal tract bymucin at the epithelial/environmental interface acting to capture salivagrowth factors. Also, combinations of growth factors, such as thosefound in saliva, may be necessary for optimal wound healing. Moreover,protease inhibitors, which are also produced by the salivary glands,appear to facilitate growth factor activity.

In addition, saliva contains adhesive proteins having protectiveproperties with regard to infection by exogenous microorganisms. Suchadhesive proteins bind exogenous microorganisms and facilitate thedegradation or expulsion thereof. From this rich source of biologicallyrelevant proteins, new secreted proteins are sought. Also, given theimportance and variety of saliva proteins, conditions involvinginadequate saliva production or secretion have inspired investigativeeffort.

Rattus norvegicus common salivary protein 1 (U00964₋₋ 1) and a murinehomolog of common salivary protein 1 (S76879₋₋ 1) have been discoveredand characterized. See, for example, Girard et al., J. Biol. Chem.268(35): 26592-601, 1993. Common salivary protein 1 is so designated asa result of the expression thereof in cells of all major salivaryglands. Evidence exists that such expression is androgen-regulated inthe rat submandibular gland. Common salivary protein 1 does not includestructural features associated with many other salivary proteins,including tandemly repeated sequences, a high density of chargedresidues and/or an unusually large proportion of a few amino acids.Common salivary protein 1 is somewhat homologous to spermine bindingproteins discussed below, but lacks the highly acidic carboxy terminaldomain thereof. Thus, the proteins may be evolutionarily related withoutbeing functionally related.

Salivary glands share significant features with other glands, such asthe prostate gland. For example, the salivary glands and prostate glandare classified as slow replicators with respect to their proliferativecapacity. See, for example, Zajicek, Med. Hypotheses 7(10): 1241-51,1981. Such slow replicators exhibit similar onotgenies and proceedduring regeneration and neoplasia through similar stages. The prostategland also appears to produce growth factors, such as EGF and NGF, andother biologically important proteins, such as kallikreins. See, forexample, Hiramatsu et al., Biochem. Int. 17(2): 311-7, 1988, Harper etal., J. Biol. Chem. 257(14): 8541-8, 1982 and Brady et al., Biochemistry28(12): 5203-10, 1988. Prostate gland function also appears to beandrogen-dependent. Consequently, proteins associated with the prostategland are also sought.

Glandular function is believed to be androgen-dependent. Expression ofsecreted glycoproteins, having spermine-binding activity, by mouse andrat prostate has also been postulated to be androgen-dependent. See, forexample, Mills et al., Nucleic Acids Res. 15: 7709-24, 1987.Spermine-binding protein mRNA expression appears to be induced byexposure to androgens, with an increase therein by 2-3 fold beingobserved within 16 hours and continuing for several days. Thus,intracellular levels of specific hormone-dependent mRNA, such asspermine binding protein mRNA, are useful markers of hormone action.See, Labrie et al., Endocrinology 124(6): 274-554, 1989.Spermine-binding protein is also useful for studying cAMP-independentprotein kinases, because the protein is under androgenic control throughthe action of such kinases. See, for example, Goueli et al., Biochem. J.(England) 230(2): 293-302, 1985.

Spermine-binding proteins bind to polyamines, such as spermine.Prostatic fluid, for example, is rich in polyamines, andspermine-binding proteins have therefore been postulated to serve ascarriers of such polyamines in the seminal fluid. Spermine-bindingproteins may also be useful to disrupt spermine-mediated pig lenssoluble protein aggregation to form cataracts. See, for example,Maekawa, Mie. Med. J. 39(2): 221-8, 1989. In addition, spermine binds toand is believed to modulate the activity of the N-methyl-D-aspartate(NMDA) receptor, a ligand-gated ion channel (Bergeron et al., J. Med.Chem. 38: 425-8, 1995). Also, spermine is believed to effect vascularsmooth muscle cell contractility via inhibition of myosin phosphatase(Sward et al., Am. J. Physiology 269(3 pt. 1): C563-71, 1995). Thus,homologs of spermine-binding proteins are sought.

The present invention provides such polypeptides for these and otheruses that should be apparent to those skilled in the art from theteachings herein.

SUMMARY OF THE INVENTION

Within one aspect the invention provides an isolated polypeptidecomprising a sequence of amino acid residues that is at least 80%identical in amino acid sequence to residues 23-178 of SEQ ID NO:2.Within one embodiment the polypeptide is at least 90% identical in aminoacid sequence to residues 23-178 of SEQ ID NO:2. Within anotherembodiment the polypeptide comprises residues 7-178 of SEQ ID NO:2.Within yet another embodiment the polypeptide comprises residues 1-178of SEQ ID NO:2. Within still another embodiment the polypeptide is atleast 1 kb in length. Within yet another embodiment the polypeptide iscovalently linked to a moiety selected from the group consisting ofaffinity tags, toxins, radionucleotides, enzymes and fluorophores.Within a related embodiment the moiety is an affinity tag selected fromthe group consisting of polyhistidine, FLAG, Glu-Glu, glutathione Stransferase and an immunoglobulin heavy chain constant region. Withinanother related embodiment the polypeptide further comprises aproteolytic cleavage site between the sequence of amino acid residuesand the affinity tag.

Within another aspect is provided an expression vector comprising thefollowing operably linked elements: a transcription promoter; a DNAsegment encoding a polypeptide as described above; and a transcriptionalterminator. Within a related embodiment the DNA segment encodes apolypeptide covalently linked to an affinity tag selected from the groupconsisting of polyhistidine, FLAG, Glu-Glu, glutathione S transferaseand an immunoglobulin heavy chain constant region. Within a relatedembodiment the DNA further encodes a secretory signal sequence operablylinked to said polypeptide. Within another embodiment the secretorysignal sequence encodes residues 7-22 of SEQ ID NO:2. Within a relatedembodiment the secretory signal sequence encodes residues 1-22 of SEQ IDNO:2. Within another related embodiment is provided a cultured cell intowhich has been introduced an expression vector as described above,wherein the cell expresses the polypeptide encoded by the DNA segment.

Within another aspect is provided a method of producing a proteincomprising: culturing a cell into which has been introduced anexpression vector as described above, whereby the cell expresses theprotein encoded by the DNA segment; and recovering the expressedprotein.

Within another aspect is provided a pharmaceutical compositioncomprising a polypeptide as described above in combination with apharmaceutically acceptable vehicle.

Within other aspects are provided an antibody that specifically binds toan epitope of a polypeptide as described above and a binding proteinthat specifically binds to an epitope of a polypeptide as describedabove.

Within a further aspect is provided an isolated polynucleotide encodinga polypeptide as described above.

Within one embodiment is provided an isolated polynucleotide asdescribed above wherein the polynucleotide is selected from the groupconsisting of, a) a sequence of nucleotides from nucleotide 168 tonucleotide 704 of SEQ ID NO:1; b) a sequence of nucleotides fromnucleotide 186 to nucleotide 704 of SEQ ID NO:2; c) a sequence ofnucleotides from nucleotide 234 to nucleotide 704 of SEQ ID NO:2; d) asequence of nucleotides from nucleotide 246 to nucleotide 704 of SEQ IDNO:2; e) allelic variants of a), b), c) or d); and

f) nucleotide sequences complementary to a), b), c), d) or e). Withinanother embodiment the polynucleotide is from 471 to 853 nucleotides inlength. Within another embodiment the polynucleotide comprisesnucleotide 1 to nucleotide 534 of SEQ ID NO:20. Within anotherembodiment the polynucleotide is DNA.

Within another aspect of the invention is provided an oligonucleotideprobe or primer comprising 14 contiguous nucleotides of a polynucleotideof SEQ ID NO:20 or a sequence complementary to SEQ ID NO:20.

Within yet another aspect is provided a method for detecting a geneticabnormality in a patient, comprising: obtaining a genetic sample from apatient; incubating the genetic sample with a polynucleotide comprisingat least 14 contiguous nucleotides of SEQ ID NO:1 or the complement ofSEQ ID NO:1, under conditions wherein said polynucleotide will hybridizeto complementary polynucleotide sequence, to produce a first reactionproduct; comparing said first reaction product to a control reactionproduct, wherein a difference between said first reaction product andsaid control reaction product is indicative of a genetic abnormality inthe patient.

Within a further aspect is provided a DNA construct encoding apolypeptide fusion, said fusion comprising a secretory signal sequenceselected from the group consisting of: (a) amino acid residues 1-22 ofSEQ ID NO:2; and (b) amino acid residues 7-22 of SEQ ID NO:2; whereinthe secretory signal sequence is operably linked to an additionalpolypeptide.

Within another aspect is provided a method for detecting zsig32polypeptides comprising: exposing a polypeptide containing sample to anantibody attached to a solid support, wherein said antibody binds to anepitope of a zsig32 polypeptide; washing said immobilizedantibody-polypeptide to remove unbound contaminants; exposing theimmobilized antibody-polypeptide to a second antibody directed to asecond epitope of a zsig32 polypeptide, wherein the second antibody isassociated with a detectable label; and detecting the detectable label.

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention and theattached drawing.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE illustrates an alignment of mouse ventral prostatespermine-binding protein (SPBP₋₋ MOUSE), Rattus norvegicus commonsalivary protein 1 (U00964₋₋ 1), a murine common salivary protein 1(S76879₋₋ 1) and a zsig32 polypeptide of the present invention. (SEQ IDNOS. 2-5)

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term "affinity tag" is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term "allelic variant" denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequence. The term allelic variant is also used herein to denote aprotein encoded by an allelic variant of a gene.

The terms "amino-terminal" and "carboxyl-terminal" are used herein todenote positions within polypeptides and proteins. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide or protein to denote proximity or relativeposition. For example, a certain sequence positioned carboxyl-terminalto a reference sequence within a protein is located proximal to thecarboxyl terminus of the reference sequence, but is not necessarily atthe carboxyl terminus of the complete protein.

The term "complement/anti-complement pair" denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term "complements of polynucleotide molecules" denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.

The term "degenerate nucleotide sequence" denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term "expression vector" denotes a DNA molecule, linear or circular,that comprises a segment encoding a polypeptide of interest operablylinked to additional segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andmay optionally include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, and the like.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term "isolated", when applied to a polynucleotide molecule, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5' and 3' untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985). When applied to a protein, the term "isolated"indicates that the protein is found in a condition other than its nativeenvironment, such as apart from blood and animal tissue. In a preferredform, the isolated protein is substantially free of other proteins,particularly other proteins of animal origin. It is preferred to providethe protein in a highly purified form, i.e., greater than 95% pure, morepreferably greater than 99% pure.

The term "operably linked", when referring to DNA segments, denotes thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator.

The term "ortholog" denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

"Paralogs" are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The term "polynucleotide" denotes a single- or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5' to the3' end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases("kb"). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term "basepairs". It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

"Probes and/or primers" as used herein can be RNA or DNA. DNA can beeither cDNA or genomic DNA. Polynucleotide probes and primers are singleor double-stranded DNA or RNA, generally synthetic oligonucleotides, butmay be generated from cloned cDNA or genomic sequences or itscomplements. Analytical probes will generally be at least 20 nucleotidesin length, although somewhat shorter probes (14-17 nucleotides) can beused. PCR primers are at least 5 nucleotides in length, preferably 15 ormore nt, more preferably 20-30 nt. Short polynucleotides can be usedwhen a small region of the gene is targeted for analysis. For grossanalysis of genes, a polynucleotide probe may comprise an entire exon ormore. Probes can be labeled to provide a detectable signal, such as withan enzyme, biotin, a radionuclide, fluorophore, chemiluminescer,paramagnetic particle and the like, which are commercially availablefrom many sources, such as Molecular Probes, Inc., Eugene, Oreg., andAmersham Corp., Arlington Heights, Ill., using techniques that are wellknown in the art.

The term "promoter" denotes a portion of a gene containing DNA sequencesthat provide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly, but not always, found inthe 5' non-coding regions of genes.

The term "receptor" denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Most nuclear receptors also exhibit amulti-domain structure, including an amino-terminal, transactivatingdomain, a DNA binding domain and a ligand binding domain. In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor).

The term "secretory signal sequence" denotes a DNA sequence that encodesa polypeptide (a "secretory peptide") that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term "splice variant" is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as "about" X or"approximately" X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery of a novel DNAsequence that encodes a polypeptide characterized by homology to mouseventral prostate spermine-binding protein (SEQ ID NO: 3). See, forexample, Mills et al., Nucleic Acids Res. 15: 7709-24, 1987. Possiblymore significant is the homology of zsig32 polypeptide to Rattusnorvegicus common salivary protein (U00964₋₋ 1; SEQ ID NO: 4), asdiscussed in Girard et al., J. Biol. Chem., 268(35): 26592-601, 1993,and murine common salivary protein 1 (S76879₋₋ 1; SEQ ID NO: 5).

In addition, the zsig32 polypeptides of the present invention preferablyincorporate one potential N-glycosylation site at amino acid 167 (Asn)of SEQ ID NO: 2. Zsig32 polypeptides of the present invention alsopreferably incorporate two potential tyrosine sulfatation sites at aminoacids 40 and 155 of SEQ ID NO: 2. Preferably, one potential proteinkinase C phosphorylation site is located at amino acid 107 (Tyr) of SEQID NO: 2. Such putative sites of phosphorylation may indicate thatzsig32, like spermine-binding protein, is regulated by cAMP-independentprotein kinases. See, for example, Goueli et al., Biochem. J. (England)230(2): 293-302, 1985. In addition, the zsig32 polypeptides of thepresent invention preferably incorporate three potential casein kinaseII phosphorylation sites at amino acids 35 (Ser), 36 (Tyr) and 107 (Tyr)of SEQ ID NO: 2. Such sites may indicate that zsig32 may be regulated bycasein kinase II. Preferably, three potential N-myristoylation sites arelocated at amino acids 46, 72 and 120 of SEQ ID NO: 2). These sites arenot 100% conserved in the aligned common salivary proteins.

Zsig32 polypeptides also preferably incorporate an adhesion motif (aminoacid residues 63-65 of SEQ ID NO: 2). According to three-dimensionalanalysis of polypeptide structure, the adhesion motif appears to beexposed. Thus, zsig32 polypeptides and fragments thereof, incorporatingsuch an adhesion motif, appear to be useful in the study of adhesion, asis more fully described herein. Also useful in this regard are fusionproteins containing zsig32 polypeptide or an exposed adhesionmotif-containing fragment thereof.

The novel polypeptides of the present invention, designated zsig32polypeptides, were initially identified by querying an EST database forsecretory signal sequences characterized by an upstream methionine startsite, a hydrophobic region of approximately 13 amino acids and acleavage site (SEQ ID NO: 6, wherein cleavage occurs between the alanineand glycine amino acid residues) in an effort to select for secretedproteins. Polypeptides corresponding to ESTs meeting those searchcriteria were compared to known sequences to identify secreted proteinshaving homology to known ligands. A single EST sequence was discoveredand predicted to be related to a secreted spermine-binding protein foundin rat ventral prostate (SPBP). See, for example, Mills et al., NucleicAcids Res. 15: 7709-24, 1987. Homology was also discovered betweenzsig32 polypeptide and Rattus norvegicus common salivary protein 1(U00964₋₋ 1), as discussed in Girard et al., J. Biol. Chem., 268(35):26592-601, 1993, and a murine common salivary protein 1 (S76879₋₋ 1).

The full sequence of the zsig32 polypeptide was obtained from a singleclone believed to contain it, wherein the clone was obtained from aparotid gland tissue library. Other libraries that might also besearched for such clones include submandibular gland, salivary gland,prostate, trachea, colon, stomach, prostate tumor, lung, thyroid, tonguetumor and the like.

Analysis of the tissue distribution of the mRNA corresponding to thisnovel DNA by Northern blot analysis using a synthetic probe (SEQ ID NO:7) showed that expression was highest in trachea, lower in prostate, andapparent but further decreased in colon and stomach. A tissuedistribution analysis by Dot blot showed extremely high expression insalivary gland, with significantly less expression in trachea andapparent but further decreased expression in prostate. In evaluatingthese results it is important to note that the Northern blot analysisdid not include the salivary gland. A single transcript size ofapproximately 1 kb was observed.

The nucleotide sequence of the N-terminal EST is described in SEQ ID NO:1, and its deduced amino acid sequence is described in SEQ ID NO: 2.Analysis of the DNA encoding a zsig32 polypeptide (SEQ ID NO: 1)revealed an open reading frame encoding either 178 (Met at amino acidnumber 1 of SEQ ID NO: 2 is the start site) or 172 amino acids (Met atamino acid number 7 of SEQ ID NO: 2 is the start site). The open readingframe comprises a signal peptide of either 22 amino acid residues(residue 1 to residue 22 of SEQ ID NO: 2; Met at position 1 is the startsite) or 16 amino acid residues (residue 7 to residue 22 of SEQ ID NO:2; Met at position 7 is the start site) and a mature polypeptide of 156amino acids (residue 23 to residue 178 of SEQ ID NO: 2). Those skilledin the art will recognize that predicted secretory signal sequencedomain boundaries are approximations based on primary sequence content,and may vary slightly; however, such estimates are generally accurate towithin ±4 amino acid residues. Therefore the present invention alsoincludes the polypeptides having amino acid sequences comprising aminoacid residues 20-178 of SEQ ID NO:2, residues 21-178 of SEQ ID NO:2,residues 22-178 of SEQ ID NO:2, residues 23-178 of SEQ ID NO:2, residues24-178 of SEQ ID NO:2, residues 25-178, residues 26-178 of SEQ ID NO:2and residues 27-178 of SEQ ID NO:2 as well as the polynucleotidesencoding them.

An alignment was prepared including zsig32 polypeptide, mouse ventralprostate spermine-binding protein SPBP₋₋ MOUSE (SEQ ID NO: 3; Met atposition 7) rat common salivary protein 1 (U00964₋₋ 1; SEQ ID NO: 4; Metat positions 1 and 7), and murine common salivary protein 1 (S76879₋₋ 1;SEQ ID NO: 5; Met at positions 1 and 7). That alignment, as shown in theFigure, revealed a block of high percent identity in the signal sequenceand a block of significant percent identity in the mature proteincorresponding to the region of SEQ ID NO: 2 from amino acid residue 7(Met), corresponding to aligned residue 7, to amino acid residue 217(Glu), corresponding to aligned residue 167. When compared, the mostconserved sequences are in the signal sequence. Also, the two commonsalivary proteins and zsig32 share a six amino acid presequence(residues 1-6 of SEQ ID NOS. 4, 5 and 6). In addition, the C-terminaltail of SPBP₋₋ MOUSE appears to be longer than that of zsig32 and to becharacterized by high asparagine/aspartic acid concentration. TheC-terminal tail of SPBP₋₋ MOUSE is believed to participate in sperminebinding. In contrast, the C-terminal tails of the salivary proteins areshorter than that of zsig32. Neither zsig32 nor the salivary proteinsincorporate a region of high asparagine/aspartic acid concentration,potentially indicating alternative regulation or specificity.

Within the region of significant identity in the mature polypeptides,the following percent identity figures are observed for the deducedamino acid sequence of zsig32 polypeptide (SEQ ID NO: 2), SPBP₋₋ MOUSE(SEQ ID NO: 3), rat common salivary protein 1, U00964₋₋ 1 (SEQ ID NO:4), and murine common salivary protein 1, S76879-1 (SEQ ID NO: 5).

    ______________________________________                                                  Zsig32  U00964.sub.-- 1                                                                         S76879.sub.-- 1                                                                      SPBP.sub.-- MOUSE                          ______________________________________                                        Zsig32    100     31        29     29                                         U00964.sub.-- 1                                                                         31      100       44     29                                         S76879.sub.-- 1                                                                         29      44        100    28                                         SPBP.sub.-- MOUSE                                                                       29      29        28     100                                        ______________________________________                                    

Highly conserved amino acids can be used as a tool to identify zsig32polypeptides or other proteins characterized by salivary gland functionindication or modulation, spermine or polyamine binding capability,cAMP-independent protein kinase- or androgen-regulation susceptibilityor the like. For instance, reverse transcription-polymerase chainreaction (RT-PCR) can be used to amplify sequences encoding a conservedmotif from RNA obtained from a variety of tissue sources. In particular,the following primers are useful for this purpose.

1) Amino acids 14-19 of SEQ ID NO: 2 (corresponding to nucleotides 40-57of SEQ ID NO: 1, nucleotides 40-57 of SEQ ID NO:20 and theircomplements);

2) Amino acids 31-36 of SEQ ID NO: 2 (corresponding to nucleotides91-108 of SEQ ID NO: 1, nucleotides 97-108 of SEQ ID NO:20 and theircomplements);

3) Amino acids 154-159 of SEQ ID NO: 2 (corresponding to nucleotides460-477 of SEQ ID NO: 1, nucleotides 460-477 of SEQ ID NO:20 and theircomplements); and

4) Amino acids 109-114 of SEQ ID NO: 2 (corresponding to nucleotides325-342 of SEQ ID NO: 1, nucleotides 325-342 of SEQ ID NO:20, and theircomplements).

The activity of polypeptides identified by such probes or ofpolypeptides encoded by polynucleotides identified by such probes can bedetermined by methods that are known in the art as generally describedherein.

Oligonucleotide probes based on the polynucleotide sequence of SEQ IDNO:1 can be used to localize the zsig32 gene to a particular chromosome.Radiation hybrid mapping is a somatic cell genetic technique developedfor constructing high-resolution, contiguous maps of mammalianchromosomes (Cox et al., Science 250:245-50, 1990). Partial or fullknowledge of a gene's sequence allows one to design PCR primers suitablefor use with chromosomal radiation hybrid mapping panels. Radiationhybrid mapping panels are commercially available which cover the entirehuman genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RHPanel (Research Genetics, Inc., Huntsville, Ala.). These panels enablerapid, PCR-based chromosomal localizations and ordering of genes,sequence-tagged sites (STSs), and other nonpolymorphic and polymorphicmarkers within a region of interest. This includes establishing directlyproportional physical distances between newly discovered genes ofinterest and previously mapped markers. The precise knowledge of agene's position can be useful for a number of purposes, including: 1)determining if a sequence is part of an existing contig and obtainingadditional surrounding genetic sequences in various forms, such as YACs,BACs or cDNA clones; 2) providing a possible candidate gene for aninheritable disease which shows linkage to the same chromosomal region;and 3) cross-referencing model organisms, such as mouse, which may aidin determining what function a particular gene might have.

Sequence tagged sites (STSs) can also be used independently forchromosomal localization. An STS is a DNA sequence that is unique in thehuman genome and can be used as a reference point for a particularchromosome or region of a chromosome. An STS is defined by a pair ofoligonucleotide primers that are used in a polymerase chain reaction tospecifically detect this site in the presence of all other genomicsequences. Since STSs are based solely on DNA sequence they can becompletely described within an electronic database, for example,Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center forBiological Information, National Institutes of Health, Bethesda, Md.http://www.ncbi.nlm. nih.gov), and can be searched with a gene sequenceof interest for the mapping data contained within these short genomiclandmark STS sequences.

The results of chromosome mapping experiments, as more fully describedin Example 3 hereof, showed that the zsig32 gene maps 7.47 cR from thetop of the human chromosome 16 linkage group on the WICGR radiationhybrid map. Relative to the centromere, its nearest proximal marker wasWI-7742 and its nearest distal maker was WI-3061. The use of surroundingmarkers positioned the zsig32 gene in the 16p13.3 region on theintegrated LDB chromosome 16 map.

The present invention also provides polynucleotide molecules, includingDNA and RNA molecules, that encode the zsig32 polypeptides disclosedherein. Those skilled in the art will readily recognize that, in view ofthe degeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NO:20 is adegenerate DNA sequence that encompasses all DNAs that encode the zsig32polypeptide of SEQ ID NO:2. Those skilled in the art will recognize thatthe degenerate sequence of SEQ ID NO:20 also provides all RNA sequencesencoding SEQ ID NO:2 by substituting U for T. Thus, zsig32polypeptide-encoding polynucleotides comprising nucleotide 61 tonucleotide 534 of SEQ ID NO:20, nucleotide 22 to nucleotide 534 of SEQID NO:20 and nucleotide 1 to nucleotide 534 of SEQ ID NO:20 and theirRNA equivalents are contemplated by the present invention. Table 1 setsforth the one-letter codes used within SEQ ID NO:20 to denote degeneratenucleotide positions. "Resolutions" are the nucleotides denoted by acode letter. "Complement" indicates the code for the complementarynucleotide(s). For example, the code Y denotes either C or T, and itscomplement R denotes A or G, A being complementary to T, and G beingcomplementary to C.

                  TABLE 1                                                         ______________________________________                                        Nucleotide Resolution                                                                             Nucleotide   Complement                                   ______________________________________                                        A          A        T            T                                            C          C        G            G                                            G          G        C            C                                            T          T        A            A                                            R          A|G                                                                           Y            C|T                                 Y          C|T                                                                           R            A|G                                 M          A|C                                                                           K            G|T                                 K          G|T                                                                           M            A|C                                 S          C|G                                                                           S            C|G                                 W          A|T                                                                           W            A|T                                 H          A|C|T                                                                D            A|G|T                      B          C|G|T                                                                V            A|C|G                      V          A|C|G                                                                B            C|G|T                      D          A|G|T                                                                H            A|C|T                      N          A|C|G|T                                                     N            A|C|G|T           ______________________________________                                    

The degenerate codons used in SEQ ID NO:20, encompassing all possiblecodons for a given amino acid, are set forth in Table 2.

                  TABLE 2                                                         ______________________________________                                               One                                                                    Amino  Letter                     Degenerate                                  Acid   Code    Codons             Codon                                       ______________________________________                                        Cys    C       TGC TGT            TGY                                         Ser    S       AGC AGT TCA TCC TCG TCT                                                                          WSN                                         Thr    T       ACA ACC ACG ACT    ACN                                         Pro    P       CCA CCC CCG CCT    CCN                                         Ala    A       GCA GCC GCG GCT    GCN                                         Gly    G       GGA GGC GGG GGT    GGN                                         Asn    N       AAC AAT            AAY                                         Asp    D       GAC GAT            GAY                                         Glu    E       GM GAG             GAR                                         Gln    Q       CAA CAG            CAR                                         His    H       CAC CAT            CAY                                         Arg    R       AGA AGG CGA CGC CGG CGT                                                                          MGN                                         Lys    K       AAA AAG            AAR                                         Met    M       ATG                ATG                                         Ile    I       ATA ATC ATT        ATH                                         Leu    L       CTA CTC CTG CTT TTA TTG                                                                          YTN                                         Val    V       GTA GTC GTG GTT    GTN                                         Phe    F       TTC TTT            TTY                                         Tyr    Y       TAC TAT            TAY                                         Trp    W       TGG                TGG                                         Ter    .       TAA TAG TGA        TRR                                         Asn|Asp                                                                     B                          RAY                                         Glu|Gln                                                                     Z                          SAR                                         Any    X                          NNN                                         ______________________________________                                    

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2. Variant sequences can be readily tested forfunctionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit "preferential codon usage." In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term "preferential codon usage" or "preferential codons" isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:20 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

Based upon homology to spermine binding protein, zsig32 polypeptides maybe used in the study of cAMP-independent protein kinases or androgens.Androgens mediate cAMP-independent protein kinase levels, which in turnmediate phosphorylation of zsig32 polypeptides. In an embodiment of thepresent invention, immobilized zsig32 polypeptides are incubated withγ-³² P-ATP and a cAMP-independent protein kinase, and incorporation of³² P into zsig32 polypeptides is monitored by known techniques.Alternatively, cells that express or are engineered to express zsig32polypeptides and that endogenously express one or more cAMP-independentprotein kinase are incubated with androgens and γ-³² P-ATP. The cellsare then lysed and incorporation of ³² P into zsig32 polypeptides ismonitored by known techniques. In addition, spermine binding proteinmRNA has been suggested as a marker for specific androgen activity, asdescribed in Labrie et al., Endocrinology 124(6): 2745-54, 1989. Zsig32polypeptide mRNA may also be useful for that purpose.

As shown in the Figure and described herein, zsig32 polypeptides alsoexhibit homology to common salivary protein 1 isolated from rat andmouse. Moreover, zsig32 polypeptides are characterized by a limitedtissue distribution. While the Northern blot and Dot blot analyses arenot in complete agreement, a high level of expression of zsig32polypeptide was observed in the salivary gland. Consequently, anotheraspect of the present invention involves the detection of zsig32polypeptides in the serum or tissue biopsy of a patient undergoingevaluation for salivary gland function or dysfunction. Such zsig32polypeptides can be detected using immunoassay techniques and antibodiescapable of recognizing zsig32 polypeptide epitopes.

More specifically, the present invention contemplates methods fordetecting zsig32 polypeptide comprising:

exposing a solution possibly containing zsig32 polypeptide to anantibody attached to a solid support, wherein said antibody binds to afirst epitope of a zsig32 polypeptide;

washing said immobilized antibody-polypeptide to remove unboundcontaminants;

exposing the immobilized antibody-polypeptide to a second antibodydirected to a second epitope of a zsig32 polypeptide, wherein the secondantibody is associated with a detectable label; and

detecting the detectable label. Serum or biopsy zsig32 polypeptideconcentration (relative to normal serum or tissue concentration) may beindicative of dysfunction of the salivary gland. Salivary glanddysfunctions include digestive dysfunction, wound healing dysfunction,inadequate saliva production or composition, mucosal integritybreakdown, failure or diminished anti-microbial function. Detection ofzsig32 polypeptide at relatively high levels in the trachea may indicatethat such polypeptides may serve as a marker of lung dysfunction.Examples of conditions associated with salivary gland or lungdysfunction include salivary gland carcinoma, sarcoidosis, pneumocysticcarinii (particularly as associated with AIDS patients), emphysema,chronic bronchitis, cystic fibrosis, ARDS, SIDS or the like. Inaddition, zsig32 polypeptides are expressed in the prostate, albeit at alower level than in the salivary gland and trachea. The prostate glandis androgen regulated and shares other properties with salivary glands.Consequently, dysfunction thereof, such as prostate adenocarcinoma orthe like, may also be detected using zsig32 polypeptides.

Also, the salivary glands synthesize and secrete a number of proteinshaving diverse biological functions. Such proteins facilitatelubrication of the oral cavity (e.g., mucins and proline-rich proteins),remineralization (e.g., statherin and ionic proline-rich proteins),digestion (e.g., amylase, lipase and proteases), provide anti-microbial(e.g., proline-rich proteins, lysozyme, histatins and lactoperoxidase)and mucosal integrity maintenance (e.g., mucins) capabilities. Inaddition, saliva is a rich source of growth factors synthesized by thesalivary glands. For example, saliva is known to contain epidermalgrowth factor (EGF), nerve growth factor (NGF), transforming growthfactor-alpha (TGF-α), transforming growth factor-beta (TGF-β), insulin,insulin-like growth factors I and II (IGF-I and IGF-II) and fibroblastgrowth factor (FGF). See, for example, Zelles et al., J. Dental. Res.74: 1826-32, 1995. Synthesis of growth factors by the salivary gland isbelieved to be androgen-dependent and to be necessary for the health ofthe oral cavity and gastrointestinal tract.

Thus, zsig32 polypeptides, agonists or antagonists thereof may betherapeutically useful for aiding digestion. To verify the presence ofthis capability in zsig32 polypeptides, agonists or antagonists of thepresent invention, such zsig32 polypeptides, agonists or antagonists areevaluated with respect to their ability to break down starch accordingto procedures known in the art. If desired, zsig32 polypeptideperformance in this regard can be compared to digestive enzymes, such asamylase, lipase, proteases and the like. In addition, zsig32polypeptides or agonists or antagonists thereof may be evaluated incombination with one or more digestive enzymes to identify synergisticeffects.

Also, zsig32 polypeptides, agonists or antagonists thereof may betherapeutically useful for promoting wound healing. To verify thepresence of this capability in zsig32 polypeptides, agonists orantagonists of the present invention, such zsig32 polypeptides, agonistsor antagonists are evaluated with respect to their ability to facilitatewound healing according to procedures known in the art. If desired,zsig32 polypeptide performance in this regard can be compared to growthfactors, such as EGF, NGF, TGF-α, TGF-β, insulin, IGF-I, IGF-II,fibroblast growth factor (FGF) and the like. In addition, zsig32polypeptides or agonists or antagonists thereof may be evaluated incombination with one or more growth factors to identify synergisticeffects.

In addition, zsig32 polypeptides, agonists or antagonists thereof may betherapeutically useful for anti-microbial applications. To verify thepresence of this capability in zsig32 polypeptides, agonists orantagonists of the present invention, such zsig32 polypeptides, agonistsor antagonists are evaluated with respect to their anti-microbialproperties according to procedures known in the art. See, for example,Barsum et al., Eur. Respir. J. 8: 709-14, 1995; Sandovsky-Losica et al.,J. Med. Vet. Mycol. (England) 28: 279-87, 1990; Mehentee et al., J. Gen.Microbiol (England) 135 (Pt. 8): 2181-8, 1989; Segal and Savage, J. Med.Vet. Mycol. 24: 477-9, 1986 and the like. If desired, zsig32 polypeptideperformance in this regard can be compared to proteins known to befunctional in this regard, such as proline-rich proteins, lysozyme,histatins, lactoperoxidase or the like. In addition, zsig32 polypeptidesor agonists or antagonists thereof may be evaluated in combination withone or more anti-microbial agents to identify synergistic effects.

Anti-microbial protective agents may be directly acting or indirectlyacting. Such agents operating via membrane association or pore formingmechanisms of action directly attach to the offending microbe.Anti-microbial agents can also act via an enzymatic mechanism, breakingdown microbial protective substances or the cell wall/membrane thereof.Anti-microbial agents, capable of inhibiting microorganism proliferationor action or of disrupting microorganism integrity by either mechanismset forth herein, are useful in methods for preventing contamination incell culture by microbes susceptible to that anti-microbial activity.Such techniques involve culturing cells in the presence of an effectiveamount of said zsig32 polypeptide or an agonist or antagonist thereof.

Also, zsig32 polypeptides or agonists thereof may be used as cellculture reagents in in vitro studies of exogenous microorganisminfection, such as bacterial, viral or fungal infection. Such moietiesmay also be used in in vivo animal models of infection. Also, themicroorganism-adherence properties of zsig32 polypeptides or agoniststhereof can be studied under a variety of conditions in binding assaysand the like.

Moreover, zsig32 polypeptides, agonists or antagonists thereof may betherapeutically useful for mucosal integrity maintenance. To verify thepresence of this capability in zsig32 polypeptides, agonists orantagonists of the present invention, such zsig32 polypeptides, agonistsor antagonists are evaluated with respect to their mucosal integritymaintenance according to procedures known in the art. See, for example,Zahm et al., Eur. Respir. J. 8: 381-6, 1995, which describes methods formeasuring viscoelastic properties and surface properties of mucous aswell as for evaluating mucous transport by cough and by ciliaryactivity. If desired, zsig32 polypeptide performance in this regard canbe compared to mucins or the like. In addition, zsig32 polypeptides oragonists or antagonists thereof may be evaluated in combination withmucins to identify synergistic effects.

In addition, a 37 amino acid peptide has been discovered which issecreted in saliva and believed to cause vasodilation of cranial bloodvessels. Such vasodilation leads to migrane attacks. This peptide isbelieved to be secreted in response to clenching of the teeth, which isalso often associated with migrane attacks. Thus, a further aspect ofthe present invention involves the determination of the vasodilatoryeffects of zsig32 polypeptides and agonists and antagonists thereof.Such determination may be made using known assay techniques. Compoundscapable of down-modulating vasodilation of blood vessels may be usefulin the prevention or treatment of migrane attacks.

Polynucleotides encoding zsig32 polypeptides are useful within genetherapy applications where it is desired to increase or inhibit zsig32activity. If a mammal has a mutated or absent zsig32 gene, the zsig32gene can be introduced into the cells of the mammal. In one embodiment,a gene encoding a zsig32 polypeptide is introduced in vivo in a viralvector. Such vectors include an attenuated or defective DNA virus, suchas, but not limited to, herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. A defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Examples of particular vectorsinclude, but are not limited to, a defective herpes simplex virus 1(HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991);an attenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al., J. Clin. Invest. 90:626-30, 1992; and adefective adeno-associated virus vector (Samulski et al., J. Virol.61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

In another embodiment, a zsig32 gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; InternationalPatent Publication NO: WO 95/07358, published Mar. 16, 1995 by Doughertyet al.; and Kuo et al., Blood 82:845, 1993. Alternatively, the vectorcan be introduced by lipofection in vivo using liposomes. Syntheticcationic lipids can be used to prepare liposomes for in vivotransfection of a gene encoding a marker (Felgner et al., Proc. Natl.Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci.USA 85:8027-31, 1988). The use of lipofection to introduce exogenousgenes into specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. More particularly, directing transfection to particularcells represents one area of benefit. For instance, directingtransfection to particular cell types would be particularly advantageousin a tissue with cellular heterogeneity, such as the pancreas, liver,kidney, and brain. Lipids may be chemically coupled to other moleculesfor the purpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

It is possible to remove the target cells from the body; to introducethe vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

Antisense methodology can be used to inhibit zsig32 gene transcription,such as to inhibit cell proliferation in vivo. Polynucleotides that arecomplementary to a segment of a zsig32-encoding polynucleotide (e.g., apolynucleotide as set froth in SEQ ID NO:1) are designed to bind tozsig32-encoding mRNA and to inhibit translation of such mRNA. Suchantisense polynucleotides are used to inhibit expression of zsig32polypeptide-encoding genes in cell culture or in a subject.

Transgenic mice, engineered to express the zsig32 gene, and mice thatexhibit a complete absence of zsig32 gene function, referred to as"knockout mice" (Snouwaert et al., Science 257:1083, 1992), may also begenerated (Lowell et al., Nature 366:740-42, 1993). These mice may beemployed to study the zsig32 gene and the protein encoded thereby in anin vivo system.

The present invention also provides reagents for use in diagnosticapplications. For example, the zsig32 gene, a probe comprising zsig32DNA or RNA, or a subsequence thereof can be used to determine if thezsig32 gene is present on chromosome 16 or if a mutation has occurred.Detectable chromosomal aberrations at the zsig32 gene locus include, butare not limited to, aneuploidy, gene copy number changes, insertions,deletions, restriction site changes and rearrangements. Theseaberrations can occur within the coding sequence, within introns, orwithin flanking sequences, including upstream promoter and regulatoryregions, and may be manifested as physical alterations within a codingsequence or changes in gene expression level.

In general, these diagnostic methods comprise the steps of (a) obtaininga genetic sample from a patient; (b) incubating the genetic sample witha polynucleotide probe or primer as disclosed above, under conditionswherein the polynucleotide will hybridize to complementarypolynucleotide sequence, to produce a first reaction product; and (iii)comparing the first reaction product to a control reaction product. Adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the patient. Geneticsamples for use within the present invention include genomic DNA, cDNA,and RNA. The polynucleotide probe or primer can be RNA or DNA, and willcomprise a portion of SEQ ID NO:1, the complement of SEQ ID NO:1, or anRNA equivalent thereof. Suitable assay methods in this regard includemolecular genetic techniques known to those in the art, such asrestriction fragment length polymorphism (RFLP) analysis, short tandemrepeat (STR) analysis employing PCR techniques, ligation chain reaction(Barany, PCR Methods and Applications 1:5-16, 1991), ribonucleaseprotection assays, and other genetic linkage analysis techniques knownin the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian,Chest 108:255-65, 1995). Ribonuclease protection assays (see, e.g.,Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA probeto a patient RNA sample, after which the reaction product (RNA-RNAhybrid) is exposed to RNase. Hybridized regions of the RNA are protectedfrom digestion. Within PCR assays, a patient's genetic sample isincubated with a pair of polynucleotide primers, and the region betweenthe primers is amplified and recovered. Changes in size or amount ofrecovered product are indicative of mutations in the patient. AnotherPCR-based technique that can be employed is single strand conformationalpolymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications1:34-8, 1991).

Within another aspect of the present invention there is provided apharmaceutical composition comprising purified zsig32 polypeptide incombination with a pharmaceutically acceptable vehicle. Suchpharmaceutical compositions may be administered to prevent or treatsalivary gland dysfunction. Such prevention or treatment may be directedto digestive dysfunction, such as a deficiency in starch breakdowncapability or efficiency, wound healing dysfunction, inadequate salivaproduction or composition or mucosal integrity breakdown. Zsig32polypeptides may also have an anti-microbial function, most likelystemming from an exposed adhesion motif as discussed herein. Also,expression of zsig32 polypeptide at a relatively high level in tracheamay indicate a role for zsig32 polypeptides in prevention or treatmentof destructive lung disease. Examples of pathological conditions,characterized by one or more of the aforementioned criteria, includexerostomia, sarcoidosis, dental caries, osteomyelitis, oral candidiasis,buccal mucosa infections, chronic inflammation (Sjogren's syndrome),mumps, chronic bronchitis, adult respiratory distress syndrome (ARDS),sudden infant death syndrome (SIDS), salivary gland carcinoma,pneumocystic carinii (particularly as associated with AIDS patients),cystic fibrosis, emphysema and the like.

Evaluation of zsig32 polypeptide involvement in such conditions may beconducted using in vivo or in vitro methods that are known to those ofordinary skill in the art. For example, bronchoalveolar lavage may beemployed in the assessment of destructive lung diseases, such aspulmonary emphysema, chronic bronchitis, cystic fibrosis, ARDS and thelike. See, for example, Luisetti et al., Respiration 59(suppl. 1): 24-7,1992. Salivary gland, lacrimal gland and labial salivary gland biopsesmay be employed in the evaluation of xerostomia. See, for example,Matsumoto et al., J. Clin. Invest. 97(8): 1969-77, 1996. This calciumchannel dependent condition has also been evaluated using fura-2 assaysof intracellular calcium ion concentration, as described in Seagrave etal., Archs. Oral Biol. 41(5): 425-30, 1996. Alymphoplasia (aly) mice area useful animal model for systemic Sjogren's syndrome, an autoimmunedisease characterized by lymphocytic infiltration into the lachrymal andsalivary glands, leading to symptomatic dry eyes and mouth. See, forexample, Furukawa et al., Brit. J. Rheum. 35: 1223-30, 1996.

The present invention provides methods for identifying agonists orantagonists of the zsig32 polypeptides disclosed herein, which agonistsor antagonists may have valuable therapeutic properties as discussedfurther herein. Within one embodiment, there is provided a method ofidentifying zsig32 polypeptide agonists, comprising providing cellsresponsive to a zsig32 polypeptide as disclosed herein, culturing thecells in the presence of a test compound and comparing the cellularresponse with the cell cultured in the presence of the zsig32polypeptide, and selecting the test compounds for which the cellularresponse is of the same type. Agonists are therefore useful to mimic oraugment the function of zsig32 polypeptides.

Within another embodiment, there is provided a method of identifyingantagonists of zsig32 polypeptide, comprising providing cells responsiveto a zsig32 polypeptide, culturing a first portion of the cells in thepresence of zsig32 polypeptide, culturing a second portion of the cellsin the presence of the zsig32 polypeptide and a test compound, anddetecting a decrease in a cellular response of the second portion of thecells as compared to the first portion of the cells. Antagonists aretherefore useful to inhibit or diminish zsig32 polypeptide function.

Zsig32 polypeptides of the present invention exhibit an adhesion motifat amino acid residues 63-65 of SEQ ID NO: 2. According tothree-dimensional analysis of polypeptide structure, the adhesion motifappears to be exposed. Thus, zsig32 polypeptides, fragments thereofincorporating such an adhesion motif or fusion proteins incorporatingzsig32 polypeptide or the adhesion motif-containing fragment thereofappear to be useful in the study of adhesion. Such study can beaccomplished using a method of modulating adhesion of platelets, forexample, in cell culture, comprising incubating platelets in a culturemedium comprising a zsig32 polypeptide as disclosed above in an amountsufficient to modulate adhesion. Assays for evaluation of plateletadhesion are known in the art.

Zsig32 polypeptides or antagonists or agonists thereof are expected beuseful in circumstances where modulation of adhesion is desired. Suchadhesion-modulating function may be used in in vitro experimentsdesigned to study adhesion, such as adhesion of microorganisms to cells,tissue or mucous. Enhancers and inhibitors of adhesion also havepotential as therapeutics for conditions requiring such modulation. Forexample, enhanced tumor cell-tumor cell adhesion in a primary solidtumor does not favor metastasis thereof. Also, diminished tumorcell-endothelial cell adhesion also does not favor metastasis formationat a site distant from the primary tumor. Assays to assess metastaticpotential, assessed using adhesion parameters, are known in the art.See, for example, Koenigsmann et al., Onkologie 17: 528-37, 1994, Asaoet al., Canc. Letts. 78: 57-62, 1994 and the like.

Preliminary analysis of mice receiving intravenous tail vein injectionsof adenovirus expressing a zsig32 polypeptides suggests that zsig32 mayincrease platelet levels 25-30% above normal. Cytokines such aserythropoietin and IL-6 also increase platelet levels by 25-30%.Therapeutic application of such activity would be useful wherever it isdesirable to increase proliferation of such cells, such as bone marrowtransplantation, in the treatment of cytopenia, such as that induced byaplastic anemia, myelodisplastic syndromes, chemotherapy or congenitalcytopenias. Application could also be made in the treatment ofthrombocytopenia. Use of proteins capable of platelet stimulation wouldalso be valuable as tools for the in vitro study of the differentiationand development of hematopoietic cells, such as for elucidating themechanisms of cell differentiation and for determining the lineages ofmature cells, and may also find utility as proliferative agents in cellculture. Platelets are directed to sites of injuries and are believed tobe mediators of wound healing and, under some circumstances, mediatorsof pathogenesis.

A still further aspect of the invention provides useful researchreagents and methods for evaluating salivary gland function. Zsig32polypeptides, agonists or antagonists thereof can be admixed with testsaliva or one or more proteins contained in saliva to provide cultureconditions under which salivary gland function can be studied. Forexample, admixture of zsig32 polypeptides, agonists or antagoniststhereof can be combined with one or more growth factors to provide aculture medium in which the wound healing properties of saliva can bestudied.

Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO: 2,SEQ ID NO: 6, SEQ ID NO: 10, or SEQ ID NO: 11, the other specific probesreferred to herein, or a sequence complementary thereto, under stringentconditions. In general, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe. Typical stringentconditions are those in which the salt concentration is less than about0.02 M at pH 7 and the temperature is at least about 60° C.

As previously noted, the isolated zsig32 polynucleotides of the presentinvention include DNA and RNA. Methods for isolating DNA and RNA arewell known in the art. It is generally preferred to isolate RNA fromsalivary gland tissues, although DNA can also be prepared using RNA fromother tissues or isolated as genomic DNA. Total RNA can be preparedusing guanidine HCl extraction followed by isolation by centrifugationin a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).Poly(A)⁺ RNA is prepared from total RNA using the method of Aviv andLeder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972). Complementary DNA(cDNA) is prepared from poly(A)⁺ RNA using known methods.Polynucleotides encoding zsig32 polypeptides are then identified andisolated by, for example, hybridization or PCR.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to mammalian, avian, amphibian, reptile, fish,insect and other vertebrate and invertebrate species. Of particularinterest are zsig32 polypeptides from other mammalian species, includingmurine, rat, porcine, ovine, bovine, canine, feline, equine and otherprimate proteins. Species homologs of the human proteins can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses the protein. Suitable sources of mRNA can be identified byprobing Northern blots with probes designed from the sequences disclosedherein. A library is then prepared from mRNA of a positive tissue ofcell line. A zsig32-encoding cDNA can then be isolated by a variety ofmethods, such as by probing with a complete or partial human cDNA orwith one or more sets of degenerate probes based on the disclosedsequences. A cDNA can also be cloned using the polymerase chainreaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primersdesigned from the sequences disclosed herein. Within an additionalmethod, the cDNA library can be used to transform or transfect hostcells, and expression of the cDNA of interest can be detected with anantibody to an epitope of a zsig32 polypeptide. Similar techniques canalso be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NO:1 and SEQ ID NO:2 represent a single allele of the humanzsig32 gene and polypeptide, and that allelic variation and alternativesplicing are expected to occur. Allelic variants can be cloned byprobing cDNA or genomic libraries from different individuals accordingto standard procedures. Allelic variants of the DNA sequence shown inSEQ ID NO: 1, including those containing silent mutations and those inwhich mutations result in amino acid sequence changes, are within thescope of the present invention, as are proteins which are allelicvariants of SEQ ID NO:2. cDNAs generated from alternatively splicedmRNAs, which retain the properties of the zsig32 polypeptide areincluded within the scope of the present invention, as are polypeptidesencoded by such cDNAs and mRNAs. Allelic variants and splice variants ofthese sequences can be cloned by probing cDNA or genomic libraries fromdifferent individuals or tissues according to standard procedures knownin the art.

The present invention also provides isolated zsig32 polypeptides thatare substantially homologous to the polypeptides of SEQ ID NO:2 andtheir orthologs. The term "substantially homologous" is used herein todenote polypeptides having 50%, preferably 60%, more preferably at least80%, sequence identity to the sequences shown in SEQ ID NO:2 or theirorthologs. Such polypeptides will more preferably be at least 90%identical, and most preferably 95% or more identical to SEQ ID NO:2 orits orthologs. Percent sequence identity is determined by conventionalmethods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16,1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9,1992. Briefly, two amino acid sequences are aligned to optimize thealignment scores using a gap opening penalty of 10, a gap extensionpenalty of 1, and the "blosum 62" scoring matrix of Henikoff andHenikoff (ibid.) as shown in Table 3 (amino acids are indicated by thestandard one-letter codes). The percent identity is then calculated as:##EQU1##

                                      TABLE 3                                     __________________________________________________________________________        R N D C Q E G HA                                                                              I L K M F P S T W Y V                                     __________________________________________________________________________    A 4                                                                           R   -1                                                                              5                                                                       N   -2                                                                              0                                                                             6                                                                       D   -2                                                                              1-2                                                                              6                                                                    C  0                                                                                -3                                                                              -3                                                                              -3                                                                            9                                                                   Q   -1                                                                             1                                                                              0  0                                                                                -3                                                                             5                                                                E   -1                                                                             0                                                                              0  2                                                                                -4                                                                             2                                                                               5                                                              G  0                                                                                -2                                                                            0   -1                                                                              -3                                                                              -2                                                                              -2                                                                            6                                                             H   -2                                                                             0                                                                              1   -1                                                                              -3                                                                             0                                                                               0                                                                                -2                                                                             8                                                          I   -1                                                                              -3                                                                              -3                                                                              -3                                                                             -1                                                                               -3                                                                              -3                                                                             -4                                                                               -3                                                                             4                                                        L   -1                                                                              -2                                                                              -3                                                                              -4                                                                              -1                                                                              -2                                                                              -3                                                                             -4                                                                               -3                                                                             2                                                                              4                                                       K   -1                                                                             2                                                                              0   -1                                                                              -3                                                                             1                                                                               1                                                                                -2                                                                              -1                                                                              -3                                                                             -2                                                                               5                                                   M   -1                                                                               -2                                                                               -3                                                                             -1                                                                              0                                                                                -2                                                                              -3                                                                              -2                                                                             1                                                                              2   -1                                                                            5                                                   F   -2                                                                              -3                                                                              -3                                                                              -3                                                                             -2                                                                               -3                                                                              -3                                                                             -3                                                                               -1                                                                             0                                                                              0   -3                                                                            0  6                                                P   -1                                                                                -2                                                                              -1                                                                              -3                                                                              -1                                                                              -1                                                                              -2                                                                              -2                                                                              -3                                                                              -3                                                                              -1                                                                              -2                                                                              -4                                                                             7                                              S  1                                                                                -1                                                                            1  0                                                                                -1                                                                             0                                                                               0                                                                              0   -1                                                                              -2                                                                              -2                                                                             0                                                                                -1                                                                              -2                                                                              -1                                                                             4                                            T  0                                                                                -1                                                                            0   -1                                                                              -1                                                                              -1                                                                              -1                                                                              -2                                                                              -2                                                                              -1                                                                              -1                                                                              -1                                                                              -1                                                                              -2                                                                              -1                                                                             1                                                                               5                                          W   -3                                                                                -4                                                                              -4                                                                              -2                                                                              -2                                                                              -3                                                                             -2                                                                               -2                                                                              -3                                                                              -2                                                                              -3                                                                             -1                                                                              1                                                                                -4                                                                              -3                                                                              -2                                                                            11                                        Y   -2                                                                                -2                                                                              -3                                                                             -2                                                                               -1                                                                              -2                                                                              -3                                                                             2                                                                                -1                                                                              -1                                                                              -2                                                                              -1                                                                             3                                                                               -3                                                                               -2                                                                              -2                                                                            2     7                                   V  0                                                                                -3                                                                              -3                                                                              -3                                                                             -1                                                                               -2                                                                              -2                                                                              -3                                                                              -3                                                                             3                                                                              1   -2                                                                            1   -1                                                                              -2                                                                              -2                                                                             0                                                                                -3  4-1                                 __________________________________________________________________________     Sequence identity of polynucleotide molecules is determined by similar     methods using a ratio as disclosed above.

Substantially homologous proteins and polypeptides are characterized ashaving one or more amino acid substitutions, deletions or additions.These changes are preferably of a minor nature, that is conservativeamino acid substitutions (see Table 4) and other substitutions that donot significantly affect the folding or activity of the protein orpolypeptide; small deletions, typically of one to about 30 amino acids;and small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue, a small linker peptide of up to about20-25 residues, or an affinity tag. Polypeptides comprising affinitytags can further comprise a proteolytic cleavage site between the zsig32polypeptide and the affinity tag. Preferred such sites include thrombincleavage sites and factor Xa cleavage sites.

                  TABLE 4                                                         ______________________________________                                        Conservative amino acid substitutions                                         ______________________________________                                        Basic:              arginine                                                                      lysine                                                                        histidine                                                 Acidic:             glutamic acid                                                                 aspartic acid                                             Polar:              glutamine                                                                     asparagine                                                Hydrophobic:        leucine                                                                       isoleucine                                                                    valine                                                    Aromatic:           phenylalanine                                                                 tryptophan                                                                    tyrosine                                                  Small:              glycine                                                                       alanine                                                                       serine                                                                        threonine                                                                     methionine                                                ______________________________________                                    

The present invention further provides a variety of other polypeptidefusions [and related multimeric proteins comprising one or morepolypeptide fusions]. For example, a zsig32 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Preferred dimerizing proteins in this regardinclude immunoglobulin constant region domains. Immunoglobulin-zsig32polypeptide fusions can be expressed in genetically engineered cells [toproduce a variety of multimeric zsig32 analogs]. Auxiliary domains canbe fused to zsig32 polypeptides to target them to specific cells,tissues, or macromolecules. For example, a zsig32 polypeptide or proteincould be targeted to a predetermined cell type by fusing a zsig32polypeptide to a ligand that specifically binds to a receptor on thesurface of the target cell. In this way, polypeptides and proteins canbe targeted for therapeutic or diagnostic purposes. A zsig32 polypeptidecan be fused to two or more moieties, such as an affinity tag forpurification and a targeting domain. Polypeptide fusions can alsocomprise one or more cleavage sites, particularly between domains. See,Tuan et al., Connect. Tiss. Res. 34:1-9, 1996.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is carried outin a cell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung etal., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.USA 90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for zsig32 amino acidresidues.

Essential amino acids in the zsig32 polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-5, 1989). In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (e.g., androgen regulation, anti-microbial activity,adhesion modulation or the like) to identify amino acid residues thatare critical to the activity of the molecule. See also, Hilton et al.,J. Biol. Chem. 271:4699-708, 1996. Sites of ligand-receptor interactioncan also be determined by physical analysis of structure, as determinedby such techniques as nuclear magnetic resonance, crystallography,electron diffraction or photoaffinity labeling, in conjunction withmutation of putative contact site amino acids. See, for example, de Voset al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol.224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. Theidentities of essential amino acids can also be inferred from analysisof homologies with related proteins.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner etal., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

Variants of the disclosed zsig32 DNA and polypeptide sequences can begenerated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid "evolution" of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

Mutagenesis methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides (e.g., those susceptible toandrogen regulation, capable of anti-microbial action or adhesionmodulation or the like) can be recovered from the host cells and rapidlysequenced using modern equipment. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

Using the methods discussed herein, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that are substantiallyhomologous to residues 23 (Gly) to 178 (Arg) of SEQ ID NO: 2 or allelicvariants thereof and retain the one or more properties of the wild-typeprotein. Such polypeptides may include additional amino acids, such asaffinity tags or the like. Such polypeptides may also include additionalpolypeptide segments as generally discussed herein.

The polypeptides of the present invention, including full-lengthproteins, fragments thereof and fusion proteins, can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly cultured cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are disclosed bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, andAusubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley and Sons, Inc., New York, 1987.

In general, a DNA sequence encoding a zsig32 polypeptide of the presentinvention is operably linked to other genetic elements required for itsexpression, generally including a transcription promoter and terminatorwithin an expression vector. The vector will also commonly contain oneor more selectable markers and one or more origins of replication,although those skilled in the art will recognize that within certainsystems selectable markers may be provided on separate vectors, andreplication of the exogenous DNA may be provided by integration into thehost cell genome. Selection of promoters, terminators, selectablemarkers, vectors and other elements is a matter of routine design withinthe level of ordinary skill in the art. Many such elements are describedin the literature and are available through commercial suppliers.

To direct a zsig32 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the zsig32 polypeptide, ormay be derived from another secreted protein (e.g., t-PA) or synthesizedde novo. The secretory signal sequence is joined to the zsig32-encodingDNA sequence in the correct reading frame. Secretory signal sequencesare commonly positioned 5' to the DNA sequence encoding the polypeptideof interest, although certain signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).Conversely, the signal sequence portion of the zsig32 polypeptide (aminoacids 1-22 or 7-22 of SEQ ID NO: 2) may be employed to direct thesecretion of an alternative protein by analogous methods.

Cultured mammalian cells are also preferred hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., eds., Current Protocols in MolecularBiology, John Wiley and Sons, Inc., New York, 1987), liposome-mediatedtransfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al.,Focus 15:80, 1993), and viral vectors (Miller and Rosman, BioTechniques7:980-90, 1989; Wang and Finer, Nat. Med. 2:714-16, 1996). Theproduction of recombinant polypeptides in cultured mammalian cells isdisclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339;Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No.4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable culturedmammalian cells include the COS-1 (ATCC NO: CRL 1650), COS-7 (ATCC NO:CRL 1651), BHK 570 (ATCC NO: CRL 10314), 293 (ATCC NO: CRL 1573; Grahamet al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g.CHO-K1; ATCC NO: CCL 61 ) cell lines. Additional suitable cell lines areknown in the art and available from public depositories such as theAmerican Type Culture Collection, Rockville, Md. In general, strongtranscription promoters are preferred, such as promoters from SV-40 orcytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitablepromoters include those from metallothionein genes (U.S. Pat. Nos.4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as "transfectants". Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as "stable transfectants." Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as "amplification." Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate.

Other drug resistance genes (e.g., hygromycin resistance, multi-drugresistance, puromycin acetyltransferase) can also be used. Alternativemarkers that introduce an altered phenotype, such as green fluorescentprotein, or cell surface proteins such as CD4, CD8, Class I MHC,placental alkaline phosphatase may be used to sort transfected cellsfrom untransfected cells by such means as FACS sorting or magnetic beadseparation technology.

Other higher eukaryotic cells can also be used as hosts, includinginsect cells, plant cells and avian cells. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.Transformation of insect cells and production of foreign polypeptidestherein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222; Bang etal., U.S. Pat. No. 4,775,624; and WIPO publication WO 94/06463. Insectcells can be infected with recombinant baculovirus, commonly derivedfrom Autographa californica nuclear polyhedrosis virus (ACNPV). DNAencoding the zsisg32 polypeptide is inserted into the baculoviral genomein place of the AcNPV polyhedrin gene coding sequence by one of twomethods. The first is the traditional method of homologous DNArecombination between wild-type AcNPV and a transfer vector containingthe zsig32 flanked by AcNPV sequences. Suitable insect cells, e.g. SF9cells, are infected with wild-type AcNPV and transfected with a transfervector comprising a zsig32 polynucleotide operably linked to an AcNPVpolyhedrin gene promoter, terminator, and flanking sequences. See, King,and Possee, The Baculovirus Expression System: A Laboratory Guide,London, Chapman & Hall; O'Reilly et al., Baculovirus Expression Vectors:A Laboratory Manual, New York, Oxford University Press., 1994; and,Richardson, Ed., Baculovirus Expression Protocols. Methods in MolecularBiology, Totowa, N.J., Humana Press, 1995. Natural recombination withinan insect cell will result in a recombinant baculovirus which containszsig32 driven by the polyhedrin promoter. Recombinant viral stocks aremade by methods commonly used in the art.

The second method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566-79, 1993). This system is sold in the Bac-to-Bac kit (LifeTechnologies, Rockville, Md.). This system utilizes a transfer vector,pFastBac1™ (Life Technologies) containing a Tn7 transposon to move theDNA encoding the zsig32 polypeptide into a baculovirus genome maintainedin E. coli as a large plasmid called a "bacmid." The pFastBac1™ transfervector utilizes the AcNPV polyhedrin promoter to drive the expression ofthe gene of interest, in this case zsig32. However, pFastBac1 ™ can bemodified to a considerable degree. The polyhedrin promoter can beremoved and substituted with the baculovirus basic protein promoter(also known as Pcor, p6.9 or MP promoter) which is expressed earlier inthe baculovirus infection, and has been shown to be advantageous forexpressing secreted proteins. See, Hill-Perkins and Possee, J. Gen.Virol. 71:971-6, 1990; Bonning et al., J. Gen. Virol. 75:1551-6, 1994;and, Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-9, 1995. In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native zsig32 secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen, Carlsbad, Calif.), or baculovirus gp67(PharMingen, San Diego, Calif.) can be used in constructs to replace thenative zsig32 secretory signal sequence. In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed zsig32 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer et al., ibid.). Using techniques knownin the art, a transfer vector containing zsig32 is transformed into E.coli, and screened for bacmids which contain an interrupted lacZ geneindicative of recombinant baculovirus. The bacmid DNA containing therecombinant baculovirus genome is isolated, using common techniques, andused to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.Recombinant virus that expresses zsig32 is subsequently produced.Recombinant viral stocks are made by methods commonly used the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF ₉₂₁ ™(Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. The cells are grown up from an inoculation density ofapproximately 2-5×10⁵ cells to a density of 1-2×10⁶ cells at which timea recombinant viral stock is added at a multiplicity of infection (MOI)of 0.1 to 10, more typically near 3. The recombinant virus-infectedcells typically produce the recombinant zsig32 polypeptide at 12-72hours post-infection and secrete it with varying efficiency into themedium. The culture is usually harvested 48 hours post-infection.Centrifugation is used to separate the cells from the medium(supernatant). The supernatant containing the zsig32 polypeptide isfiltered through micropore filters, usually 0.45 μm pore size.Procedures used are generally described in available laboratory manuals(King and Possee, ibid.; O'Reilly, et al., ibid.; Richardson, ibid.).Subsequent purification of the zsig32 polypeptide from the supernatantcan be achieved using methods described herein.

Fungal cells, including yeast cells, and particularly cells of the genusSaccharomyces, can also be used within the present invention, such asfor producing zsig32 polypeptide fragments or polypeptide fusions.Methods for transforming yeast cells with exogenous DNA and producingrecombinant polypeptides therefrom are disclosed by, for example,Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No.4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No.5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cellsare selected by phenotype determined by the selectable marker, commonlydrug resistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A preferred vector system for use in yeast isthe POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No.4,931,373), which allows transformed cells to be selected by growth inglucose-containing media. Suitable promoters and terminators for use inyeast include those from glycolytic enzyme genes (see, e.g., Kawasaki,U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; andBitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. Seealso U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986 andCregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (τ) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a zsig32polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-cultured transferred into the host cell. P.methanolica cells are cultured in a medium comprising adequate sourcesof carbon, nitrogen and trace nutrients at a temperature of about 25° C.to 35° C. Liquid cultures are provided with sufficient aeration byconventional means, such as shaking of small flasks or sparging offermentors. A preferred culture medium for P. methanolica is YEPD (2%D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1%Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006%L-leucine).

Expressed recombinant zsig32 polypeptides (or chimeric zsig32polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,Piscataway, N.J.) being particularly preferred. Exemplarychromatographic media include those media derivatized with phenyl,butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated byexploitation of their structural properties. For example, immobilizedmetal ion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (Methodsin Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, Glu-Glu, animmunoglobulin domain) may be constructed to facilitate purification.

Protein refolding (and optionally reoxidation) procedures may beadvantageously used. It is preferred to purify the protein to >80%purity, more preferably to >90% purity, even more preferably >95%, andparticularly preferred is a pharmaceutically pure state, that is greaterthan 99.9% pure with respect to contaminating macromolecules,particularly other proteins and nucleic acids, and free of infectiousand pyrogenic agents. Preferably, a purified protein is substantiallyfree of other proteins, particularly other proteins of animal origin.

Zsig32 polypeptides or fragments thereof may also be prepared throughchemical synthesis. Zsig32 polypeptides may be monomers or multimers;glycosylated or non-glycosylated; pegylated or non-pegylated; and may ormay not include an initial methionine amino acid residue.

A zsig32 polypeptide binding protein can also be used in purificationapplications. The binding protein is immobilized on a solid support,such as beads of agarose, cross-linked agarose, glass, cellulosicresins, silica-based resins, polystyrene, cross-linked polyacrylamide,or like materials that are stable under the conditions of use. Methodsfor linking polypeptides to solid supports are known in the art, andinclude amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, and hydrazide activation. The resulting medium willgenerally be configured in the form of a column, and fluids containingligand are passed through the column one or more times to allow ligand(e.g., zsig32 polypeptide) to bind to the receptor/binding protein. Theligand is then eluted using changes in salt concentration, chaotropicagents (guanidine HCl), or pH to disrupt ligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument (BIAcore™,Pharmacia Biosensor, Piscataway, N.J.) may be advantageously employed.Such receptor, antibody, member of a complement/anti-complement pair orfragment is immobilized onto the surface of a receptor chip. Use of thisinstrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40,1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. Areceptor, antibody, member or fragment is covalently attached, usingamine or sulfhydryl chemistry, to dextran fibers that are attached togold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

Zsig32 can be measured in vitro using cultured cells or in vivo byadministering molecules of the claimed invention to the appropriateanimal model. For instance, zsig32 transfected (or co-transfected)expression host cells may be embedded in an alginate environment andinjected (implanted) into recipient animals. Alginatepoly-L-lysinemicroencapsulation, permselective membrane encapsulation and diffusionchambers have been described as a means to entrap transfected mammaliancells or primary mammalian cells. These types of non-immunogenic"encapsulations" or microenvironments permit the transfer of nutrientsinto the microenvironment, and also permit the diffusion of proteins andother macromolecules secreted or released by the captured cells acrossthe environmental barrier to the recipient animal. Most importantly, thecapsules or microenvironments mask and shield the foreign, embeddedcells from the recipient animal's immune response. Suchmicroenvironments can extend the life of the injected cells from a fewhours or days (naked cells) to several weeks (embedded cells).

Alginate threads provide a simple and quick means for generatingembedded cells. The materials needed to generate the alginate threadsare readily available and relatively inexpensive. Once made, thealginate threads are relatively strong and durable, both in vitro and,based on data obtained using the threads, in vivo. The alginate threadsare easily manipulable and the methodology is scalable for preparationof numerous threads. In an exemplary procedure, 3% alginate is preparedin sterile H₂ O, and sterile filtered. Just prior to preparation ofalginate threads, the alginate solution is again filtered. Anapproximately 50% cell suspension (containing about 5×10⁵ to about 5×10⁷cells/ml) is mixed with the 3% alginate solution. One ml of thealginate/cell suspension is extruded into a 100 mM sterile filteredCaCl₂ solution over a time period of .sup.˜ 15 min, forming a "thread".The extruded thread is then transferred into a solution of 50 mM CaCl₂,and then into a solution of 25 mM CaCl₂. The thread is then rinsed withdeionized water before coating the thread by incubating in a 0.01%solution of poly-L-lysine. Finally, the thread is rinsed with LactatedRinger's Solution and drawn from solution into a syringe barrel (withoutneedle attached). A large bore needle is then attached to the syringe,and the thread is intraperitoneally injected into a recipient in aminimal volume of the Lactated Ringer's Solution.

An alternative in vivo approach for assaying proteins of the presentinvention involves viral delivery systems. Exemplary viruses for thispurpose include adenovirus, herpesvirus, vaccinia virus andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for a review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997). The adenovirus system offers several advantages:adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with a large number of available vectors containingdifferent promoters. Also, because adenoviruses are stable in thebloodstream, they can be administered by intravenous injection.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential E1 gene has been deleted from the viral vector, and the viruswill not replicate unless the E1 gene is provided by the host cell (thehuman 293 cell line is exemplary). When intravenously administered tointact animals, adenovirus primarily targets the liver. If theadenoviral delivery system has an E1 gene deletion, the virus cannotreplicate in the host cells. However, the host's tissue (e.g., liver)will express and process (and, if a secretory signal sequence ispresent, secrete) the heterologous protein. Secreted proteins will enterthe circulation in the highly vascularized liver, and effects on theinfected animal can be determined.

The adenovirus system can also be used for protein production in vitro.By culturing adenovirus-infected non-293 cells under conditions wherethe cells are not rapidly dividing, the cells can produce proteins forextended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293S cells can be grown in suspension cultureat relatively high cell density to produce significant amounts ofprotein (see Garnier et al., Cytotechnol. 15:145-55, 1994). With eitherprotocol, an expressed, secreted heterologous protein can be repeatedlyisolated from the cell culture supernatant. Within the infected 293Scell production protocol, non-secreted proteins may also be effectivelyobtained. Zsig32 polypeptides can also be used to prepare antibodiesthat specifically bind to zsig32 epitopes, peptides or polypeptides.Antibodies generated from this immune response can be isolated andpurified as described herein. Methods for preparing and isolatingpolyclonal and monoclonal antibodies are well known in the art. See, forexample, Current Protocols in Immunology, Cooligan, et al. (eds.),National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrooket al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982.

As would be evident to one of ordinary skill in the art, polyclonalantibodies can be generated from inoculating a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, hamsters, guinea pigs and rats as well as transgenic animals suchas transgenic sheep, cows, goats or pigs. Antibodies may also beexpressed in yeast and fungi in modified forms as well as in mammalianand insect cells. The zsig32 polypeptide or a fragment thereof serves asan antigen (immunogen) to inoculate an animal or elicit an immuneresponse. Suitable antigens would include the zsig32 polypeptide encodedby SEQ ID NO:2 from amino acid residue 21-175 of SEQ ID NO:2, or acontiguous 9-175 amino acid residue fragment thereof. The immunogenicityof a zsig32 polypeptide may be increased through the use of an adjuvant,such as alum (aluminum hydroxide) or Freund's complete or incompleteadjuvant. Polypeptides useful for immunization also include fusionpolypeptides, such as fusions of zsig32 or a portion thereof with animmunoglobulin polypeptide or with maltose binding protein. Thepolypeptide immunogen may be a full-length molecule or a portionthereof. If the polypeptide portion is "hapten-like", such portion maybe advantageously joined or linked to a macromolecular carrier (such askeyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanustoxoid) for immunization.

As used herein, the term "antibodies" includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab')₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally"cloaking" them with a human-like surface by replacement of exposedresidues, wherein the result is a "veneered" antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Human antibodies can also be madein mice having a humanized humoral immune system (Mendez et al., Nat.Genet. 14:146-56, 1997).

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to zsig32 protein orpeptide, and selection of antibody display libraries, in phage orsimilar vectors (for instance, through use of immobilized or labeledzsig32 protein or peptide). Mutagenesis methods discussed herein, inparticular domain shuffling, can be used to generate and matureantibodies.

The antibodies of the current invention, or fragments thereof, can beused to direct molecules to a specific target. For example, as T-bodies,chimeric receptors combining antibody recognition with T cell effectorfunction, (Eshhar et al., Springer Semin Immunopathol. 18:199-209, 1996;Eshhar, Cancer Immunol. Immunother. 45:131-6, 1997). Intrabodies,engineered single-chain antibodies expressed inside the cell and havinghigh affinity and specificity for intracellular targets. Such moleculeshave use in gene therapy and treatment of infectious diseases (Marasco,Immunotechnology 1:1-19, 1995; Marasco et al., Gene Ther. 4:11-5, 1997;Rondon and Marasco, Annu. Rev. Microbiol. 51:257-83, 1997 and Mhashilkaret al., J. Virol. 71:6486-94, 1997). Diabodies, bispecific non-covalentdimers of scFv antibodies useful for immunodiagnosis andtherapeutically. In addition they can be constructed in bacteria(Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993).

Antibodies herein specifically bind if they bind to a zsig32polypeptide, peptide or epitope with a binding affinity (K_(a)) of 10⁶M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹or greater, and most preferably 10⁹ M⁻¹ or greater. The binding affinityof an antibody can be readily determined by one of ordinary skill in theart, for example, by Scatchard analysis (Scatchard, ibid.).

Genes encoding polypeptides having potential zsig32 polypeptide bindingdomains, "binding proteins", can be obtained by screening random ordirected peptide libraries displayed on phage (phage display) or onbacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. Alternatively,constrained phage display libraries can also be produced. These peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andpeptide display libraries and kits for screening such libraries areavailable commercially, for instance from Clontech (Palo Alto, Calif.),Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly,Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Peptidedisplay libraries can be screened using the zsig32 sequences disclosedherein to identify proteins which bind to zsig32. These "bindingproteins" which interact with zsig32 polypeptides can be usedessentially like an antibody, for tagging cells; for isolating homologpolypeptides by affinity purification; directly or indirectly conjugatedto drugs, toxins, radionuclides and the like. These binding proteins canalso be used in analytical methods such as for screening expressionlibraries and neutralizing activity. The binding proteins can also beused for diagnostic assays for determining circulating levels ofpolypeptides; for detecting or quantitating soluble polypeptides asmarker of underlying pathology or disease. To increase the half-life ofthese binding proteins, they can be conjugated. Their biologicalproperties may be modified by dimerizing or multimerizing for use asagonists or antagonists.

A variety of assays known to those skilled in the art can be utilized todetect antibodies and/or binding proteins which specifically bind tozsig32 proteins or peptides. Exemplary assays are described in detail inAntibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold SpringHarbor Laboratory Press, 1988. Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radioimmunoassay,radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA),dot blot or Western blot assay, inhibition or competition assay, andsandwich assay. In addition, antibodies can be screened for binding towild-type versus mutant zsig32 protein or polypeptide.

Antibodies and binding proteins to zsig32 may be used for tagging cellsthat express zsig32; for isolating zsig32 by affinity purification; fordiagnostic assays for determining circulating levels of zsig32polypeptides; for detecting or quantitating soluble zsig32 as marker ofunderlying pathology or disease; in analytical methods employing FACS;for screening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockzsig32 polypeptide adhesion modulating or anti-microbial or likeactivity in vitro and in vivo. Suitable direct tags or labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmarkers, chemiluminescent markers, magnetic particles and the like;indirect tags or labels may feature use of biotin-avidin or othercomplement/anti-complement pairs as intermediates. Antibodies herein mayalso be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to zsig32or fragments thereof may be used in vitro to detect denatured zsig32 orfragments thereof in assays, for example, Western Blots or other assaysknown in the art.

In another embodiment, polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the polypeptide has multiple functional domains (i.e.,an activation domain or a ligand binding domain, plus a targetingdomain), a fusion protein including only the targeting domain may besuitable for directing a detectable molecule, a cytotoxic molecule or acomplementary molecule to a cell or tissue type of interest. Ininstances where the domain only fusion protein includes a complementarymolecule, the anti-complementary molecule can be conjugated to adetectable or cytotoxic molecule. Such domain-complementary moleculefusion proteins thus represent a generic targeting vehicle forcell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates. Thebioactive polypeptide or antibody conjugates described herein can bedelivered intravenously, intraarterially, intraductally with DMSO,intramuscularly, subcutaneously, intraperitoneally, also by transdermalmethods, by electro-transfer, orally or via inhalant.

Molecules of the present invention can be used to identify and isolatereceptors involved in salivary gland function or saliva composition. Forexample, proteins and peptides of the present invention can beimmobilized on a column and membrane preparations run over the column(Immobilized Affinity Ligand Techniques, Hermanson et al., eds.,Academic Press, San Diego, Calif., 1992, pp.195-202). Proteins andpeptides can also be radiolabeled (Methods in Enzymol., vol. 182, "Guideto Protein Purification", M. Deutscher, ed., Acad. Press, San Diego,1990, 721-737) or photoaffinity labeled (Brunner et al., Ann. Rev.Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol.33:1167-1180, 1984) and specific cell-surface proteins can beidentified.

For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Intravenous administrationwill be by bolus injection or infusion over a typical period of one toseveral hours. In general, pharmaceutical formulations will include azsig32 protein in combination with a pharmaceutically acceptablevehicle, such as saline, buffered saline, 5% dextrose in water or thelike. Formulations may further include one or more excipients,preservatives, solubilizers, buffering agents, albumin to preventprotein loss on vial surfaces, etc. Methods of formulation are wellknown in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Therapeutic doses will generally determinedby the clinician according to accepted standards, taking into accountthe nature and severity of the condition to be treated, patient traits,etc. Determination of dose is within the level of ordinary skill in theart. The proteins may be administered for acute treatment, over one weekor less, often over a period of one to three days or may be used inchronic treatment, over several months or years.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Extension of EST Sequence

The novel zsig32 polypeptide-encoding polynucleotides of the presentinvention were initially identified by querying an EST database forsecretory signal sequences characterized by an upstream methionine startsite, a hydrophobic region of approximately 13 amino acids and acleavage site (SEQ ID NO: 6, wherein cleavage occurs between the alanineand glycine amino acid residues) in an effort to select for secretedproteins. Polypeptides corresponding to ESTs meeting those searchcriteria were compared to known sequences to identify secreted proteinshaving homology to known ligands. A single EST sequence was discoveredand predicted to be related to a mouse ventral prostate spermine-bindingprotein (SPBP). See, for example, Mills et al. cited above. To identifythe corresponding cDNA, a clone considered likely to contain the entirecoding region was used for sequencing. Using an Invitrogen S.N.A.P.™Miniprep kit (Invitrogen, Corp., San Diego, Calif.) according tomanufacturer's instructions a 5 ml overnight culture in LB+50 μg/mlampicillin was prepared. The template was sequenced on an ABIPRISM™model 377 DNA sequencer (Perkin-Elmer Cetus, Norwalk, Conn.) using theABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit(Perkin-Elmer Corp.) according to manufacturer's instructions.Oligonucleotides ZC694 (SEQ ID NO: 8), ZC695 (SEQ ID NO: 9) to the T7and SP6 promoters on the clone-containing vector were used as sequencingprimers. Oligonucleotides ZC13183 (SEQ ID NO: 10), ZC13187 (SEQ ID NO:11) were used to complete the sequence from the clone. Sequencingreactions were carried out in a Hybaid OmniGene Temperature CyclingSystem (National Labnet Co. SEQUENCHER™ 3.0 sequence analysis software(Gene Codes Corporation, Ann Arbor, Mich.) was used for data analysis.The resulting 853 bp sequence is disclosed in SEQ ID NO: 1.

Example 2 Tissue Distribution

Northerns were performed using Human Multiple Tissue Blots from Clontech(Palo Alto, Calif.). A 40 bp DNA probe (ZC12493; SEQ ID NO: 7) to the 5'end of the oligonucleotide sequence of the mature polypeptide shown inSEQ ID NO: 1 was radioactively labeled with P using T4 polynucleotidekinase and forward reaction buffer (GIBCO BRL, Gaithersburg, Md.)according to the manufacturer's specifications. The probe was purifiedusing a NUCTRAP push column (Stratagene Cloning Systems, La Jolla,Calif.). EXPRESSHYB (Clontech, Palo Alto, Calif.) solution was used forprehybridization and as a hybridizing solution for the Northern blots.Hybridization took place overnight at 42° C., and the blots were thenwashed in 2× SSC and 0.05 SDS at RT, followed by a wash in 1× SSC and0.1% SDS at 71° C. One transcript size was observed at approximately 650bp. Signal intensity was highest for prostate, stomach and trachea, withrelatively less intense signals in spleen and colon.

A RNA Master Dot Blot (Clontech) that contained RNAs from varioustissues that were normalized to 8 housekeeping genes was also probedwith the 40 bp DNA probe (SEQ ID NO: 7). The blot was prehybridized andthen hybridized overnight with 10⁶ cpm/ml of the probe at 42° C.,according to the manufacturer's specifications. The blot was washed with2× SSC and 0.05% SDS at RT, followed by a wash in 0.1× SSC and 0.1% SDSat 71° C. After a 48 hour exposure, highest expression was seen in thesalivary gland, with much weaker signals in trachea and still weakersignals in prostate. Note that in prostate and trachea, a 2 kb band wasalso observed.

Example 3 Chromosomal Assignment and Placement of zsig32

Zsig32 was mapped to chromosome 16 using the commercially availableGeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc.,Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panel containsPCRable DNAs from each of 93 radiation hybrid clones, plus two controlDNAs (the HFL donor and the A23 recipient). A publicly available WWWserver (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allowsmapping relative to the Whitehead Institute/MIT Center for GenomeResearch's radiation hybrid map of the human genome (the "WICGR"radiation hybrid map) which was constructed with the GeneBridge 4Radiation Hybrid Panel.

For the mapping of zsig32 with the GeneBridge 4 RH Panel, 20 μlreactions were set up in a 96-well microtiter plate (Stratagene) andused in a RoboCycler Gradient 96 thermal cycler (Stratagene). Each ofthe 95 PCR reactions consisted of 2 μl 10× KlenTaq PCR reaction buffer(Clontech), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,Calif.), 1 μl sense primer, ZC 13,703 (SEQ ID NO:21), 1 μl antisenseprimer, ZC 13,704 (SEQ ID NO:22), 2 μl RediLoad (Research Genetics,Inc.), 0.4 μl 50× Advantage KlenTaq Polymerase Mix (ClontechLaboratories, Inc.), 25 ng of DNA from an individual hybrid clone orcontrol and ddH₂ O for a total volume of 20 μl. The reactions wereoverlaid with an equal amount of mineral oil and sealed. The PCR cyclerconditions were as follows: an initial 1 cycle 5 minute denaturation at95° C., 35 cycles of a 1 minute denaturation at 95° C., 1 minuteannealing at 60° C. and 1.5 minute extension at 72° C., followed by afinal 1 cycle extension of 7 minutes at 72° C. The reactions wereseparated by electrophoresis on a 2% agarose gel (Life Technologies,Gaithersburg, Md.).

The results showed that Zsig32 maps 7.47 cR₋₋ 3000 from the frameworkmarker WI-7742 on the WICGR chromosome 16 radiation hybrid map. Proximaland distal framework markers were WI-7742 (D16S2960) and WI-3061(D16S2965), respectively. The use of surrounding markers positionsZsig32 in the 16p13.3 region on the integrated LDB chromosome 16 map(The Genetic Location Database, University of Southhampton, www server:http://cedar.genetics. soton.ac.uk/public₋₋ html/).

Example 4 Creation of Mammalian Expression Vectors zsig32NF/PZP9,zsig32CF/pZP9 and zsig32/pZP9

Three expression vectors were prepared for the zsig32 polypeptide,zSIG32CF/pZP9 and zSIG32NF/pZP9, wherein the constructs are designed toexpress a zsig25 polypeptide with a C- or N-terminal FLAG tag (SEQ IDNO: 16) and zSIG32/pZP9 expressing untagged zsig32 polypeptides.

ZSIG32/pZP9

A approximately 800 bp restriction digest fragment of ZSIG-32 DNA wasderived from the clone described in Example 1 above. Ten microliters ofthe clone was digested with 1.5 μl each of the restriction enzymes EcoRI and Not I. The resultant ligation fragment was then run on a 0.8% LMPagarose gel (Seaplaque GTG) with 0.5× TBE buffer. A band of thepredicted size was excised and the DNA was purified from the gel with aQIAQUICK® column (Qiagen) according the manufacturer's instructions.

The excised, restriction digested zsig32 DNA was subcloned into plasmidpZP9 which had been cut with Eco RI and Not I. Plasmid CF/pZP9(deposited at the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md.) is a mammalian expression vector containing anexpression cassette having the mouse metallothionein-1 promoter,multiple restriction sites for insertion of coding sequences, a stopcodon and a human growth hormone terminator. The plasmid also has an E.coli origin of replication, a mammalian selectable marker expressionunit having an SV40 promoter, enhancer and origin of replication, a DHFRgene and the SV40 terminator. zSIG25CF/pZP9

A 553 bp PCR generated ZSIG-32 DNA fragment was created using ZC13465(SEQ ID NO:23) and ZC13447 (SEQ ID NO:26) as PCR primers and thetemplate described in Example 1 above. The PCR reaction was incubated at94° C. for 5 minutes, and then run for 10 cycles of 30 seconds at 94° C.and 2 minutes at 72° C., followed by 15 cycles at 94° C. for 30 secondsand 65° C. for 2 minutes. The resultant PCR product was then run on a0.9% GTG/TBE agarose gel with 1× TBE buffer. A band of the predictedsize was excised and the DNA was purified from the gel with a QIAQUICK™column (Qiagen) according the manufacturer's instructions. The DNA wasdigested with the restriction enzymes BAM HI (Boehringer Mannheim) andEco RI (Gibco BRL), followed by phenol/chloroform/isoamyl alcoholextraction and ETOH/glycogen precipitated.

The excised, restriction digested zsig32 DNA was subcloned into plasmidCF/pZP9 which had been cut with Eco RI and Bam HI. The zSIG32/CFpZP9expression vector uses the native zSIG32 signal peptide, and the FLAGepitope (SEQ ID NO:16) is attached at the C-terminus as a purificationaid. Plasmid CF/pZP9 (deposited at the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md.) is a mammalian expression vectorcontaining an expression cassette having the mouse metallothionein-1promoter, multiple restriction sites for insertion of coding sequences,a sequence encoding the FLAG tag (SEQ ID NO:16), a stop codon and ahuman growth hormone terminator. The plasmid also has an E. coli originof replication, a mammalian selectable marker expression unit having anSV40 promoter, enhancer and origin of replication, a DHFR gene and theSV40 terminator.

zSIG25NF/pZP9

A 490 bp PCR generated zSIG32/NF DNA fragment was created in accordancewith the procedure set forth above using Z13448 (SEQ ID NO:25) andZC13449 (SEQ ID NO:26) as PCR primers. The purified PCR fragment wasdigested with the restriction enzymes Bam HI (Boehringer Mannheim) andXho I (Gibco BRL), followed by phenol/chloroform/isoamyl alcoholextraction and ETOH/glycogen precipitation.

The excised and restriction digested zSIG32 DNA was subcloned intoplasmid NF/pZP9 which had been cut with Bam HI and Xba I. ThezSIG32/NFpZP9 expression vector incorporates the TPA leader and attachesthe FLAG tag (SEQ ID NO:16) to the N-terminal of the zsig25polypeptide-encoding polynucleotide sequence. Plasmid NF/pZP9 (depositedat the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md.) is a mammalian expression vector containing anexpression cassette having the mouse metallothionein-1 promoter, a TPAleader peptide followed by the sequence encoding the FLAG tag (SEQ IDNO:16), multiple restriction sites for insertion of coding sequences,and a human growth hormone terminator. The plasmid also contains an E.coli origin of replication, a mammalian selectable marker expressionunit having an SV40 promoter, enhancer and origin of replication, a DHFRgene and the SV40 terminator.

For the untagged zsig32 construct 100 ng of the zsig32 insert and 90 ngof the Eco RI/Not I digested pZP9 vector were ligated as described forthe tagged constructs. For the N- and C-tagged constructs, 10 ng of therestriction digested inserts and 20 ng of the corresponding vectors wereligated at room temperature for 4 hours. One microliter of each ligationreaction was independently electroporated into DH10B competent cells(GIBCO BRL, Gaithersburg, Md.) according to manufacturer's direction andplated onto LB plates containing 50 mg/ml ampicillin, and incubatedovernight. Colonies were screened by PCR as described above. Forzsig32/pZP9 screens the primers were ZC6583 (SEQ ID NO:27) and ZC5020(SEQ ID NO:28), for zSIG32CF/pZP9 screens the primers were, ZC13465 (SEQID NO:23) and ZC13447 (SEQ ID NO:24) and for zSIG32NF/pZP9 screens theprimers were ZC13448 (SEQ ID NO:25) and ZC13449 (SEQ ID NO:26). Theinsert sequence of positive clones, 1000 bp for zsig32 untagged, 490 bpfragment for zSIG32NF and a 553 bp fragment for zSIG32/CF were verifiedby sequence analysis. A large scale plasmid preparation was done using aQIAGEN™ Maxi prep kit (Qiagen) according to manufacturer's instructions.

Example 5 Mammalian Expression of zsig32

BHK 570 cells (ATCC NO: CRL-10314) were plated in 10 cm tissue culturedishes and allowed to grow to approximately 50 to 70% confluencyovernight at 37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL HighGlucose, (Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum (Hyclone,Logan, Utah), 1 μM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 μMsodium pyruvate (Gibco BRL)). The cells were then transfected with theplasmid zsig32NF/pZP9 (N-terminal FLAG tag), zsig32CF/pZP9 (C-terminalFLAG tag), or zsig32/pZP9 (untagged), using Lipofectamine™ (Gibco BRL),in serum free (SF) media formulation (DMEM, 10 mg/ml transferrin, 5mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).Sixteen micrograms of zsig32NF/pZP9 and 16 μg of zsig32CF/pZP9 wereseparately diluted into 15 ml tubes to a total final volume of 640 μlwith SF media. In separate tubes, 35 μl of Lipofectamine (Gibco BRL) wasmixed with 605 μl of SF medium. The Lipofectamine™ mix was added to theDNA mix and allowed to incubate approximately 30 minutes at roomtemperature. Five milliliters of SF media was added to theDNA:Lipofectamine™ mixture. Three plates of cells were rinsed once with5 ml of SF media, aspirated, and the DNA:Lipofectamine™ mixture wasadded. The cells were incubated at 37° C. for five hours, then 6.4 ml ofDMEM/10% FBS, 1% PSN media was added to each plate. The plates wereincubated at 37° C. overnight and the DNA:Lipofectamine™ mixture wasreplaced with fresh FBS/DMEM media the next day. On day 2post-transfection, the cells were split into the selection media(DMEM/FBS media from above with the addition of 1 μM methotrexate (SigmaChemical Co., St. Louis, Mo.)) in 150 mm plates at 1:10, 1:20 and 1:50.The cells were refed at day 5 post-transfection with fresh selectionmedia. Approximately 12 days post-transfection, two 150 mm culturedishes of methotrexate resistant colonies from each transfection weretrypsinized and the cells were pooled and plated into a T-162 flask andtransferred to large scale culture.

Example 6 Large Scale Mammalian Expression of zsig32

One T-162 flask, containing confluent cells expressing zsig32/NF and oneflask containing zsig32/CF expressing cells, obtained from theexpression procedure described above, were expanded into five T-162flasks. One of the five resulting flasks was used to freeze down fourcryovials, and the other four flasks were used to generate a Nunc cellfactory.

The cells from the four T-165 flasks of zsig32/NF and zsig32/CF werecombined and used to seed two Nunc cell factories (10 layers,commercially available from VWR). Briefly, the cells from the T-162flasks described above were detached using trypsin, pooled, and added to1.5 liters ESTEP1 media (668.7 g/50 L DMEM (Gibco), 5.5 g/50 L pyruvicacid, sodium salt 96% (Mallinckrodt), 185.0 g/50 L NaHCO₃ (Mallinkrodt),5.0 mg/ml and 25 ml/50 L insulin (JRH Biosciences), 10.0 mg/ml and 25ml/50 L transferrin (JRH Biosciences), 2.5 L/50 L fetal bovine serum(characterized) (Hyclone), 1 μM MTX, with pH adjusted to 7.05±0.05)prewarmed to 37° C. The media containing the cells was then poured intothe Nunc cell factories via a funnel. The cell factories were placed ina 37° C./5.0% CO2 incubator.

At 80-100% confluence, a visual contamination test (phenol red colorchange) was performed on the Nunc cell factories. Since no contaminationwas observed, supernatant from the confluent factories was poured into asmall harvest container, sampled and discarded. The adherent cells werethen washed once with 400 ml PBS. To detach the cells from thefactories, 100 mls of trypsin was added to each and removed and thecells were then incubated for 5 to 10 minutes in the residual trypsin.The cells were collected following two, 200 ml washes of ESTEP1 media.To each of ten ESTEP1 media-containing bottles (1.5 liters each, at 37°C.) was added 40 mls of collected cells. One 1.5 liter bottle was thenused to fill one Nunc factory. Each cell factory was placed in a 37°C./5.0% CO₂ incubator.

At 80-90% confluence, a visual contamination test (phenol red colorchange) was performed on the Nunc cell factories. Since no contaminationwas observed, supernatant from the confluent factories was poured into asmall harvest container, sampled and discarded. Cells were then washedonce with 400 ml PBS. 1.5 liters of ESTEP2 media (668.7 g/50 L DMEM(Gibco), 5.5 g/50 L pyruvic acid, sodium salt 96% (Mallinckrodt), 185.0g/50 L NaHCO₃ (Mallinkrodt), 5.0 mg/ml, 25 ml/50 L insulin, 10.0 mg/mland 25 ml/50 L transferrin) was added to each Nunc cell factory. Thecell factories were incubated at 37° C./5.0% CO₂.

At approximately 48 hours a visual contamination test (phenol red colorchange) was performed on the Nunc cell factories. Supernatant from eachfactory was poured into small harvest containers. A total of 13.5 literswas collected from all 10 factories. Fresh serum-free media (1.5 liters)was poured into each Nunc cell factory, and the factories were incubatedat 37° C./5.0 CO₂. One ml of supernatant harvest was transferred to amicroscope slide, and subjected to microscopic analysis forcontamination. The contents of the small harvest containers for eachfactory were pooled and immediately filtered. A second harvest was thenperformed, substantially as described above at 44 hours (13.5 L wereobtained) and the cell factories were discarded thereafter. Anaseptically assembled filter train apparatus was used for asepticfiltration of the harvest supernatant (conditioned media). Assembly wasa follows: tubing was wire-tied to an Opti-Cap filter (Millipore Corp.,Bedford, Mass.) and a Gelman Supercap 50 filter (Gelman Sciences, AnnArbor, Mich.). The Supercap 50 filter was also attached to a sterilecapped container located in a hood; tubing located upstream of theMillipore Opti-cap filter was inserted into a peristaltic pump; and thefree end of the tubing was placed in the large harvest container. Theperistaltic pump was run between 200 and 300 rpm, until all of theconditioned media passed through the 0.22 μm final filter into a sterilecollection container. The filtrate was placed in a 4° C. cold roompending purification. The media was concentrated 10× with a Millipore 5kDA cut off concentrator (Millipore Corp., Bedford, Mass.) according tomanufacturer's direction and subjected to Western Blot analysis using ananti-FLAG tag antibody (Kodak).

Example 7 Purification of Mammalian Expressed FLAG-tagged zsig32Polypeptides

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used to purify zsig32 constructs having anN-terminal or C-terminal flag tag. Protein was purified from the culturemedium of a mixture of baby hamster kidney cell (BHK) clones thatproduced the N- or C-terminal tagged protein (zsig32NF and zsig32CF). Atotal of 25 liters of conditioned media from BHK cells was sequentiallysterile filtered through a 4 inch, 0.2 mM Millipore (Bedford, Mass.)OptiCap capsule filter and a 0.2 mM Gelman (Ann Arbor, Mich.) Supercap50. The material was then concentrated to about 1.3 liters using anAmicon (Beverly, Mass.) DC 10 L concentrator fitted with an A/G Tech(Needham, Mass.) hollow fiber cartridge with a 15 sq. ft. 3000 kDacutoff membrane. The concentrated material was again sterile-filteredwith the Gelman filter as described above. A 25.0 ml sample of anti-FlagSepharose (Eastman Kodak, Rochester, N.Y.) was added to the sample forbatch adsorption and the mixture was gently agitated on a Wheaton(Millville, N.J.) roller culture apparatus for 18.0 h at 4° C.

The mixture was then poured into a 5.0×20.0 cm Econo-Column (Bio-Rad,Laboratories, Hercules, Calif.) and the gel was washed with 30 columnvolumes of phosphate buffered saline (PBS). The unretained flow-throughfraction was discarded. Once the absorbance of the effluent at 280 nMwas less than 0.05, flow through the column was reduced to zero and theanti-Flag Sepharose gel was washed with 2.0 column volumes of PBScontaining 0.2 mg/ml of Flag peptide,N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C (SEQ ID NO:16) (Eastman Kodak).After 1.0 hour at 4° C., flow was resumed and the eluted protein wascollected. This fraction is referred to as the peptide elution. Theanti-Flag Sepharose gel was washed with 2.0 column volumes of 0.1Mglycine, pH 2.5, and the glycine wash was collected separately. The pHof the glycine-eluted fraction was adjusted to 7.0 by the addition of asmall volume of 10× PBS and stored at 4° C. for future analysis ifneeded.

The peptide elution was concentrated to 5.0 ml using a 5,000 molecularweight cutoff membrane concentrator (Millipore, Bedford, Mass.)according to the manufacturer's instructions. The concentrated peptideelution was then separated from free peptide by chromatography on a1.5×50 cm Sephadex G-50 (Pharmacia, Piscataway, N.J.) columnequilibrated in PBS at a flow rate of 1.0 ml/min using a BioCad SprintHPLC system (PerSeptive BioSystems, Framingham, Mass.). Two-ml fractionswere collected and the absorbance at 280 nM was monitored. The firstpeak of material absorbing at 280 nM and eluting near the void volume ofthe column was collected.

By SDS-PAGE and Western analysis with anti-Flag M2 antibodies (Kodak)the purified zsig32 NF/CF preparation was composed of one majorCoomassie Blue-stained band of apparent molecular weight 25,000. Twominor bands of apparent molecular weights 22,000 and 27,000, were alsoobserved on the Coomassie Blue-stained gel. All three bands showedcross-reactivity with the anti-Flag M2 antibody on Western blots.Migration of the proteins on SDS-PAGE gels and Western blots was notchanged by reducing agents.

The protein concentration of the purified proteins (0.67 mg/ml) wasdetermined by BCA analysis (Pierce, Rockford, Ill.) and the material wasaliquoted, and stored at -80° C.

Example 8 Creation of Baculovirus Expression Vectors pFSG32, pFSGE32,pSSGE32 and pLSGE32

Four expression vectors were prepared to express zsig32 polypeptides ininsect cells: pFSG32, designed to express an untagged zsig32polypeptide; and pFSGE32, pSSGE32 and pLSGE32, designed to express azsig32 polypeptide with a C-terminal Glu-Glu tag (SEQ ID NO:12).

pFSG32

A 559 bp PCR generated zsig32 DNA fragment was created using ZC13404(SEQ ID NO:13) and ZC13409 (SEQ ID NO:14) as PCR primers andzsig32/pZP9, described above, as a template. The PCR reaction wasincubated at 94° C. for 2 minutes, followed by 30 cycles of 45 secondsat 94° C., 1 minute at 60° C. and 72° C. for 1 minute with a 1second/cycle segment extension. The resultant PCR product was then runon a 3% gel (2% NuSieve/1% BRL agarose). The 559 bp fragment wascaptured by diluting 15 fold with 0.1 mM EDTA pH 8.0 and then ligatedinto the vector pCR2.1 (TA Cloning Kit, Invitrogen Inc., San Diego,Calif.) according to manufacturer's instructions. The resultant cloneswere screened for the proper insert orientation and sequenced to confirmidentity. The resulting clone, designated pSG32a, was digested with BamHI and Asp718 and the digest run on a 1% SeaPlaque/1% NuSieve agarosegel. The band was excised, diluted to 0.5% agarose with 2 mM MgCl₂,melted at 65° C. and ligated into a Bam HI/Asp718 digested baculovirusexpression vector, pFastBac1 (Bac-to-Bac™ System, GIBCO-BRL,Gaithersburg, Md.). Forty four nanograms of the restriction digestedzsig32 insert and 126 ng of the corresponding vector were ligatedovernight. The ligation mix was diluted 3 fold in TE (10 mM Tris-HCl, pH7.5 and 1 mM EDTA) and 4 fmol of the diluted ligation mix wastransformed into DH5α Library Efficiency competent cells (LifeTechnologies) according to manufacturer's direction by heat shock for 45seconds in a 42° C. waterbath. The ligated DNA was diluted in 450 μSOCmedia (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) and plated onto LBplates containing 100 μg/ml ampicillin. The plates were incubatedovernight at 37° C. Plasmid DNA was prepared using the QiaVac Miniprep8system (Qiagen) according the manufacturer's directions. The clones werescreened by restriction digest with Hind III/BspE1.

pFSGE32

A zsig32 fragment having a C-terminal Glu-Glu tag (SEQ ID NO:12) wasgenerated by PCR as described above using oligonucleotide primersZC13404 (SEQ ID NO:13) and ZC13407 (SEQ ID NO:15). A fragment of theexpected size, 580 bp, was detected by gel electrophoresis. The DNAfragment was digested with the restriction enzymes Bam HI and Asp718 andthe resulting 565 bp zsig32 restriction fragment was ligated into a BamHI/Asp718 digested pFastBac1 vector and transformed into DH10a cells asdescribed above.

pSSGE32 and pLSGE32

A 580 bp zsig32 fragment having a C-terminal Glu-Glu tag (SEQ ID NO:12)was generated by PCR as described above. The fragment was visualized bygel electrophoresis, restriction digested and ligated into theexpression vectors, pFBPL2 and pFBPS2, as described above. The vectors,pFBPL2 and pFBPS2, were derived from a modified pFastBac1™ vector. Thepolyhedrin promoter was removed and substituted with a short (FBPS) orlong (FBPL) version of the baculovirus basic protein promoter as isknown in the art (Hill-Perkins and Possee, ibid.; Bonning et al., ibid.;and, Chazenbalk and Rapoport, ibid.).

One microliter of each of the above constructs was used to independentlytransform 20 μl DH10Bac Max Efficiency competent cells (GIBCO-BRL,Gaithersburg, Md.) according to manufacturer's instruction, by heatshock at 42° C. for 45 seconds. The transformants were then diluted inan appropriate volume of SOC media and plated on to Luria Agar platescontaining 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/mltetracycline, IPTG and Bluo Gal. The cells were incubated for 48 hoursat 370° C. A color selection was used to identify those cells havingvirus that had incorporated into the plasmid (referred to as a"bacmid"). Those colonies, which were white in color, were picked foranalysis. Bacmid DNA was isolated from positive colonies and screenedfor the correct insert using PCR. Oligonucleotide primers ZC976 (SEQ IDNO:17) and ZC 447 (SEQ ID NO:18) were used and those having the correctinsert were used to transfect Spodoptera frugiperda (Sf9) cells.

Sf9 cells were seeded at 5×10⁶ cells per 35 mm plate and allowed toattach for 1 hour at 27° C. Five microliters of bacmid DNA was dilutedwith 100 μl Sf-900 II SFM. Six to 10 μl of CellFECTIN Reagent (LifeTechnologies) was diluted with 100 μl Sf-900 II SMF. The bacmid DNA andlipid solutions were gently mixed and incubated 30-45 minutes at roomtemperature. The media from one plate of cells were aspirated, and thelipid-DNA mixture to which 0.8 ml of Sf-900 II SFM was added. The cellswere incubated at 27° C. for 4-5 hours, then 2 ml of Sf-900 II mediacontaining penicillin/streptomycin was added to each plate. The plateswere incubated at 27° C., 90% humidity, for 72 hours after which thevirus was harvested.

Primary Amplification

Sf9 cells were grown in 50 ml Sf-900 II SFM in a 50 ml shake flask to anapproximate density of 0.04-0.50×10⁶ cells/ml. They were thentransfected with 50-1000 μl of the virus stock from above and incubatedat 27° C. for 35 days after which time the virus was harvested, titer0.53×10⁸ pfu/ml. To scale up, 1.5×10⁶ SF9 cells/ml were added to fiveliters of SF-900 II SFM and grown for 91 hours. The cells were thentransfected with the harvested virus (MOI 0.2) and incubated as abovefor 71 hours.

Example 9 Purification of Baculovirus Expressed Glu-Glu-tagged zsig32polypeptides

Unless otherwise noted, all operations were carried out at 4° C. Amixture of protease inhibitors were added to a 2 liter sample ofconditioned media from C-terminal Glu-Glu (EE) tagged zsig32baculovirus-infected Sf9 cells to final concentrations of 2.5 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis,Mo.), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, Ind.),0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). The sample was centrifuged at 10,000 rpm for 30min at 4° C. in a Beckman JLA-10.5 rotor (Beckman Instruments) in aBeckman Avanti J25I centrifuge (Beckman Instruments) to remove celldebris. To the supernatant fraction was added a 50.0 ml sample ofanti-EE Sepharose, prepared as described below, and the mixture wasgently agitated on a Wheaton (Millville, N.J.) roller culture apparatusfor 18.0 h at 4° C.

The mixture was poured into a 5.0×20.0 cm Econo-Column (Bio-RadLaboratories) and the gel was washed with 30 column volumes of phosphatebuffered saline (PBS). The unretained flow-through fraction wasdiscarded. Once the absorbance of the effluent at 280 nM was less than0.05, flow through the column was reduced to zero and the anti-EESepharose gel was washed with 2.0 column volumes of PBS containing 0.2mg/ml of EE peptide (AnaSpec, San Jose, Calif.). The peptide used hasthe sequence Glu-Tyr-Met-Pro-Val-Asp (SEQ ID NO: 19). After 1.0 hour at4° C., flow was resumed and the eluted protein was collected. Thisfraction was referred to as the peptide elution. The anti-EE Sepharosegel was washed with 2.0 column volumes of 0.1 M glycine, pH 2.5, and theglycine wash was collected separately. The pH of the glycine-elutedfraction was adjusted to 7.0 by the addition of a small volume of 10×PBS and stored at 4° C.

The peptide elution was concentrated to 5.0 ml using a 5,000 molecularweight cutoff membrane concentrator (Millipore) according to themanufacturer's instructions. The concentrated peptide elution wasseparated from free peptide by chromatography on a 1.5×50 cm SephadexG-50 (Pharmacia) column equilibrated in PBS at a flow rate of 1.0 ml/minusing a BioCad Sprint HPLC (PerSeptive BioSystems). Two ml fractionswere collected and the absorbance at 280 nM was monitored. The firstpeak of material absorbing at 280 nM and eluting near the void volume ofthe column was collected. This material represented purified zsig32CEEand was composed of two major bands of apparent molecular weights 19,000and 24,000 on Coomassie Blue-stained SDS-PAGE gels. These bands werepresent in about equimolar amounts. Both bands showed cross-reactivitywith anti-EE antibodies by Western blotting of the purified material.The protein concentration (0.53 mg/ml) of the purified proteins wasdetermined by BCA analysis (Pierce) and the material was aliquoted, andstored at -80° C.

Preparation of anti-EE Sepharose

A 100 ml bed volume of protein G-Sepharose (Pharmacia) was washed 3times with 100 ml of PBS containing 0.02% sodium azide using a 500 mlNalgene 0.45 micron filter unit. The gel was washed with 6.0 volumes of200 mM triethanolamine, pH 8.2 (TEA, Sigma), and an equal volume of EEantibody solution containing 900 mg of antibody was added. After anovernight incubation at 4° C., unbound antibody was removed by washingthe resin with 5 volumes of 200 mM TEA as described above. The resin wasresuspended in 2 volumes of TEA, transferred to a suitable container,and dimethylpimilimidate-2 HCl (Pierce), dissolved in TEA, was added toa final concentration of 36 mg/ml of gel. The gel was rocked at roomtemperature for 45 min and the liquid was removed using the filter unitas described above. Nonspecific sites on the gel were then blocked byincubating for 10 minutes at room temperature with 5 volumes of 20 mMethanolamine in 200 mM TEA. The gel was then washed with 5 volumes ofPBS containing 0.02% sodium azide and stored in this solution at 4° C.

Example 9 Construction of zsig32 Amino Terminal FLAG-Tagged PichiaExpression Vector

Expression of zsig32 in Pichia methanolica utilizes the expressionsystem described in co-assigned WIPO publication WO 97/17450. Anexpression plasmid containing all or part of a polynucleotide encodingzsig32 was constructed via homologous recombination. The expressionvector was built from pCZR190, which contains the AUG1 promoter,followed by the alpha factor prepro (αFpp) leader sequence, followed byan amino-terminal FLAG tag (NF), a blunt-ended Sma I restriction sitefor insertion of the gene sequence of interest, a translational stopcodon, followed by the AUG1 terminator, the ADE2 selectable marker, andfinally the AUG1 3' untranslated region. Also included in this vectorare the URA3 and CEN-ARS sequences required for selection andreplication in S. cerevisiae, and the AmpR and colE1 ori sequencesrequired for selection and replication in E. coli. The zsig32 sequenceinserted into this vector begins at residue 22 (Ala) of the zsig32 aminoacid sequence (SEQ ID NO:2).

Two construct specific linkers were prepared and along with zsig32, werehomologously recombined into the yeast expression vector pCZR190. TheN-terminal linker comprises 70 base pairs of the αFpp coding sequencejoined to a nucleotide sequence encoding a FLAG tag (SEQ ID NO:16)followed by 70 base pairs of nucleotide sequence encoding a portion ofthe amino-terminus from the mature zsig32 sequence. The C-terminallinker comprises about 70 base pairs of carboxy terminus coding sequenceof the zsig32 joined with 70 base pairs of AUG1 terminator sequence.

The N-terminal linker was synthesized by a PCR reaction. Briefly, to afinal reaction volume of 100 μl was added 1 pm each of each linkerZC13735 (SEQ ID NO:29), ZC14291 (SEQ ID NO:30), and 100 pmol of eachprimer ZC13497 (SEQ ID NO:31) and ZC14279 (SEQ ID NO:32), 10 μl of 10×PCR buffer (Boehringer Mannheim), 1 μl Two polymerase (BoehringerMannheim), 10 μl of 0.25 mM nucleotide triphosphate mix (Perkin Elmer)and dH₂ O. The PCR reaction was incubated at 94° C. for 1.5 minutes,followed by 10 cycles of 30 seconds at 94° C., 1 minute at 50° C. and 1minute at 72° C., concluded with a 10 minute extension at 72°. Theresulting 138 bp double stranded, NF-tagged linker is disclosed in SEQID NO:33.

The C-terminal untagged zsig32 linker was made via a PCR reaction asdescribed using oligonucleotides linkers ZC14346 (SEQ ID NO:34) andZC14218 (SEQ ID NO:35) and primers ZC14278 (SEQ ID NO:36) and ZC13734(SEQ ID NO:37). The resulting 153 bp double stranded, C-terminaluntagged linker is disclosed in SEQ ID NO:38.

The NF-zsig32 plasmid was made by homologously recombining 100 ng of SmaI digested pCZR202 acceptor vector, the 1 μg of Eco RI-Xho I zsig32 cDNAdonor fragment, 1 μg of N-terminal FLAG-tagged zsig32 linker (SEQ IDNO:33) and 1 μg of C-terminal linker (SEQ ID NO:38) into S. cerevisiae.One hundred microliters of competent yeast cells (S. cerevisiae) wascombined with 10 μl of each of the fragments and linkers and transferredto a 0.2 cm electroporation cuvette. The yeast/DNA mixture waselectropulsed at 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To the cuvette wasadded 600 μl of 1.2 M sorbitol and 300 μl aliquots of the yeast/sorbitolmixture was plated onto two URA D plates and incubated at 30° C.

After about 48 hours the Ura⁺ yeast transformants from a single platewere resuspended in 2.5 ml H₂ O and spun briefly to pellet the yeastcells. The cell pellet was resuspended in 1 ml of lysis buffer (2%Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Fivehundred microliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube and theDNA precipitated with 600 μl ethanol (EtOH), followed by a 10 minutes at4° C. The DNA pellet was resuspended in 100 μl H₂ O.

Five microliters of the resuspended DNA prep was used to transform 40 μlof electrocompetent E. coli cells (DH10B, Gibco BRL). The cells wereelectropulsed at 2.0 kV and 400 ohms. Following electroporation, 1 mlSOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) was added and the cells were allowed to recover for 1 hour at37° C. prior to plating 250 μl aliquots on four LB AMP plates (LB broth(Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression construct wereidentified by PCR screening. The primers used to amplify the N-taggedzsig32 clone were ZC13479 (SEQ ID NO:31) and ZC13734 (SEQ ID NO:37). Theinsert sequence of positive clones, identified by a 625 bp fragment,were verified by sequence analysis. One such clone was designatedpGMN12-1. Larger scale plasmid DNA was isolated using Qiagen maxi kits(Qiagen) and the DNA was digested with Not I to liberate thePichia-zsig32 expression cassette from the vector backbone. The Not IDNA fragment was then transformed into the Pichia methanolica expressionhost, PMAD16. This was done by mixing 100 μl of prepared competentPMAD16 cells with 10 μg of Not I digested NF-tagged zsig32 fragment andtransferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixturewas electropulsed at 0.75 kV, 25 μF, infinite ohms. To the cuvette wasadded 1 ml of 1× Yeast Nitrogen Base and 500 μl aliquots were platedonto two ADE DS (0.056%-Ade-Trp-Thr powder, 0.67% yeast nitrogen basewithout amino acids, 2% D-glucose, 0.5% 200× tryptophan, threoninesolution, and 18.22% D-sorbitol) plates for selection and incubated at30° C. The resulting NF-tagged-zsig32 plasmid was designatedPMAD16::pGMN12-1. Transformants were then picked and screened viaWestern blot for high-level NF-tagged zsig32 expression and subjected tolarge scale fermentation.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 38                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 853 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Coding Se - #quence                                             (B) LOCATION: 168...701                                                       (D) OTHER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - GAATTCGGCT CGAGAGGAAG AGCCCCACGG CCAGCTCCTT CCTGTTCCCC TG - #GCGGCCCC         60                                                                          - TCGCTTCTTC CTTCTGGATG GGGGCCCAGG GGGCCCAGGA GAGTATAAAG GC - #GATGTGGA        120                                                                          #CAC CGG      176ACCAGA CGCCCAGTCA CAGGCGAGAG CCCTGGG ATG                     #His Arg         Met                                                          #                 1                                                           - CCA GAG GCC ATG CTG CTG CTG CTC ACG CTT GC - #C CTC CTG GGG GGC CCC          224                                                                          Pro Glu Ala Met Leu Leu Leu Leu Thr Leu Al - #a Leu Leu Gly Gly Pro           #     15                                                                      - ACC TGG GCA GGG AAG ATG TAT GGC CCT GGA GG - #A GGC AAG TAT TTC AGC          272                                                                          Thr Trp Ala Gly Lys Met Tyr Gly Pro Gly Gl - #y Gly Lys Tyr Phe Ser           #35                                                                           - ACC ACT GAA GAC TAC GAC CAT GAA ATC ACA GG - #G CTG CGG GTG TCT GTA          320                                                                          Thr Thr Glu Asp Tyr Asp His Glu Ile Thr Gl - #y Leu Arg Val Ser Val           #                50                                                           - GGT CTT CTC CTG GTG AAA AGT GTC CAG GTG AA - #A CTT GGA GAC TCC TGG          368                                                                          Gly Leu Leu Leu Val Lys Ser Val Gln Val Ly - #s Leu Gly Asp Ser Trp           #            65                                                               - GAC GTG AAA CTG GGA GCC TTA GGT GGG AAT AC - #C CAG GAA GTC ACC CTG          416                                                                          Asp Val Lys Leu Gly Ala Leu Gly Gly Asn Th - #r Gln Glu Val Thr Leu           #        80                                                                   - CAG CCA GGC GAA TAC ATC ACA AAA GTC TTT GT - #C GCC TTC CAA GCT TTC          464                                                                          Gln Pro Gly Glu Tyr Ile Thr Lys Val Phe Va - #l Ala Phe Gln Ala Phe           #    95                                                                       - CTC CGG GGT ATG GTC ATG TAC ACC AGC AAG GA - #C CGC TAT TTC TAT TTT          512                                                                          Leu Arg Gly Met Val Met Tyr Thr Ser Lys As - #p Arg Tyr Phe Tyr Phe           100                 1 - #05                 1 - #10                 1 -       #15                                                                           - GGG AAG CTT GAT GGC CAG ATC TCC TCT GCC TA - #C CCC AGC CAA GAG GGG          560                                                                          Gly Lys Leu Asp Gly Gln Ile Ser Ser Ala Ty - #r Pro Ser Gln Glu Gly           #               130                                                           - CAG GTG CTG GTG GGC ATC TAT GGC CAG TAT CA - #A CTC CTT GGC ATC AAG          608                                                                          Gln Val Leu Val Gly Ile Tyr Gly Gln Tyr Gl - #n Leu Leu Gly Ile Lys           #           145                                                               - AGC ATT GGC TTT GAA TGG AAT TAT CCA CTA GA - #G GAG CCG ACC ACT GAG          656                                                                          Ser Ile Gly Phe Glu Trp Asn Tyr Pro Leu Gl - #u Glu Pro Thr Thr Glu           #       160                                                                   - CCA CCA GTT AAT CTC ACA TAC TCA GCA AAC TC - #A CCC GTG GGT CGC TAGGG        706                                                                          Pro Pro Val Asn Leu Thr Tyr Ser Ala Asn Se - #r Pro Val Gly Arg               #   175                                                                       - TGGGGTATGG GGCCATCCGA GCTGAGGCCA TCTGTGTGGT GGTGGCTGAT GG - #TACTGGAG        766                                                                          - TAACTGAGTC GGGACGCTGA ATCTGAATCC ACCAATAAAT AAAGCTTCTG CA - #GAATCAGT        826                                                                          #            853   AGGG CGGCCGC                                               - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 178 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met His Arg Pro Glu Ala Met Leu Leu Leu Le - #u Thr Leu Ala Leu Leu         #                15                                                           - Gly Gly Pro Thr Trp Ala Gly Lys Met Tyr Gl - #y Pro Gly Gly Gly Lys         #            30                                                               - Tyr Phe Ser Thr Thr Glu Asp Tyr Asp His Gl - #u Ile Thr Gly Leu Arg         #        45                                                                   - Val Ser Val Gly Leu Leu Leu Val Lys Ser Va - #l Gln Val Lys Leu Gly         #    60                                                                       - Asp Ser Trp Asp Val Lys Leu Gly Ala Leu Gl - #y Gly Asn Thr Gln Glu         #80                                                                           - Val Thr Leu Gln Pro Gly Glu Tyr Ile Thr Ly - #s Val Phe Val Ala Phe         #                95                                                           - Gln Ala Phe Leu Arg Gly Met Val Met Tyr Th - #r Ser Lys Asp Arg Tyr         #           110                                                               - Phe Tyr Phe Gly Lys Leu Asp Gly Gln Ile Se - #r Ser Ala Tyr Pro Ser         #       125                                                                   - Gln Glu Gly Gln Val Leu Val Gly Ile Tyr Gl - #y Gln Tyr Gln Leu Leu         #   140                                                                       - Gly Ile Lys Ser Ile Gly Phe Glu Trp Asn Ty - #r Pro Leu Glu Glu Pro         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Thr Glu Pro Pro Val Asn Leu Thr Tyr Se - #r Ala Asn Ser Pro Val         #               175                                                           - Gly Arg                                                                     - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 199 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Met Leu Leu Leu Leu Thr Leu Ala Phe Leu Al - #a Ser Pro Thr Cys Arg         #                15                                                           - Ala Gln Asn Val Leu Gly Asn Ala Ala Gly Ly - #s Tyr Phe Tyr Val Gln         #            30                                                               - Gly Glu Asp Gln Gly Gln Leu Lys Gly Met Ar - #g Ile Phe Leu Ser Val         #        45                                                                   - Phe Lys Phe Ile Lys Gly Phe Gln Leu Gln Ph - #e Gly Ser Asn Trp Thr         #    60                                                                       - Asp Val Tyr Gly Thr Arg Ser Asp Asn Phe Il - #e Asp Phe Leu Leu Glu         #80                                                                           - Asp Gly Glu His Val Ile Lys Val Glu Gly Se - #r Ala Val Ile Cys Leu         #                95                                                           - Thr Ser Leu Thr Phe Thr Thr Asn Lys Gly Ar - #g Val Ala Thr Phe Gly         #           110                                                               - Val Arg Arg Gly Arg Tyr Phe Ser Asp Thr Gl - #y Gly Ser Asp Lys His         #       125                                                                   - Leu Val Thr Val Asn Gly Met His Ala Pro Gl - #y Leu Cys Val Arg Gly         #   140                                                                       - Ile Gly Phe Lys Trp Gly Asn Ile Asn Ala As - #n Gly Asn Asp His Tyr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asn Asn Lys Glu Asp Lys Ala Asp Asn Lys As - #p Ala Asp Asn Lys Asp         #               175                                                           - Ala Asp Asn Lys Asp Asp Gly Asp Glu Asp As - #p Asp Gly Asn Asp Asp         #           190                                                               - Asp Asp Gln Lys Asp Glu Ser                                                         195                                                                   - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 166 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Leu Pro Gln Leu Glu Ala Met Leu Pro Le - #u Leu Ile Leu Ala Phe         #                15                                                           - Leu Gly Thr Pro Ala Val Leu Thr Gln Ser Ar - #g Tyr His Gly Ser Glu         #            30                                                               - Thr Gly Lys His Phe Cys Ile Val Ala Pro Gl - #u Gly Glu Pro Val Thr         #        45                                                                   - Gly Ile Trp Ala Ser Leu Lys Asn Asn Ile Le - #u Ser Ser Ile Arg Leu         #    60                                                                       - Lys Phe Gly Asn Asn Trp Ser Gln Glu Tyr Gl - #y Ser Ser Gly Arg Ala         #80                                                                           - Glu Ile Glu Val Lys Leu Asn Pro Asp Glu Th - #r Val Leu Gly Phe Ser         #                95                                                           - Gly Ser Phe Tyr Ile Phe Met His Gln Ile Il - #e Ile Thr Thr Ser Gln         #           110                                                               - Pro Arg Glu Leu Ile Ile Gly Pro Leu Thr Gl - #y Arg Tyr Val Tyr Thr         #       125                                                                   - Ser Tyr Pro Glu Asn Pro Asn His Val Phe Ar - #g Gly Ile Cys Gly Tyr         #   140                                                                       - Tyr Val Thr Gly Gly Leu Lys Gly Met Arg Ty - #r Leu Trp Gly Asn Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asn Gly Thr Cys Thr Glu                                                                     165                                                           - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 170 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Met Phe Gln Leu Glu Ala Met Leu Pro Leu Le - #u Ile Leu Ala Phe Leu         #                15                                                           - Gly Thr Pro Thr Val Leu Thr Gln Asp Tyr Hi - #s Gly Pro Glu Val Gly         #            30                                                               - Lys His Ser Cys Thr Ser Ala Pro Glu Gly Ly - #s Asn Ile Thr Ser Ile         #        45                                                                   - Arg Val Phe Leu Gln Gly Arg Ser Ile Val Gl - #y Ile Gln Phe Asn Tyr         #    60                                                                       - Asn Asn Glu Asp Gly Gln Val Tyr Gly Ser Th - #r Ala Gly Lys Val Met         #80                                                                           - Val Ala Arg Leu Asn Asn Glu Glu Ser Ile Il - #e Ala Ala Glu Gly Thr         #                95                                                           - Tyr Ser Pro Ser Ala Leu Thr Gln Ile Ile Ph - #e Thr Thr Asn Gln Pro         #           110                                                               - Arg Gln Leu Met Val Gly Tyr Tyr Val Gly Se - #r Ser Glu Tyr Ser Ser         #       125                                                                   - Phe Pro Asp Asp Pro Ser His Val Leu Lys Gl - #y Ala Cys Val Ser Trp         #   140                                                                       - Arg Ala Gly Gly Ile Lys Ser Ile Leu Phe Le - #u Trp Gly Thr Glu Asn         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Ser Cys Val Lys Tyr Gly His Ser Gly                                     #               170                                                           - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Leu Leu Thr Leu Ala Leu Leu Gly Gly Pro Th - #r Trp Ala Gly Lys             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other                                               -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC12493                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #    40            CCTC CTCCAGGGCC ATACATCTTC                                 - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other                                               -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC694                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 # 20               AGGG                                                       - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other                                               -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC695                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 # 19               TAG                                                        - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other                                               -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC13183                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                # 20               TTGC                                                       - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other                                               -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC13187                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                # 20               ATCA                                                       - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Glu Glu Tyr Met Pro Met Glu                                                  1               5                                                            - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC13404                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #     38           TGCA CCGGCCAGAG GCCATGCT                                   - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC13409                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #        34        CACG GGTGAGTTTG CTGA                                       - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 55 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE:  ZC13407                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - CCGGTACCTA TTCCATCGGC ATGTATTCTT CGCGACCCAC GGGTGAGTTT GC - #TGA              55                                                                          - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Asp Tyr Lys Asp Asp Asp Asp Lys                                              1               5                                                            - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC976                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #  18              CC                                                         - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE:  ZC447                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Glu Tyr Met Pro Val Asp                                                      1               5                                                            - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 534 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - ATGCAYMGNC CNGARGCNAT GYTNYTNYTN YTNACNYTNG CNYTNYTNGG NG - #GNCCNACN         60                                                                          - TGGGCNGGNA ARATGTAYGG NCCNGGNGGN GGNAARTAYT TYWSNACNAC NG - #ARGAYTAY        120                                                                          - GAYCAYGARA THACNGGNYT NMGNGTNWSN GTNGGNYTNY TNYTNGTNAA RW - #SNGTNCAR        180                                                                          - GTNAARYTNG GNGAYWSNTG GGAYGTNAAR YTNGGNGCNY TNGGNGGNAA YA - #CNCARGAR        240                                                                          - GTNACNYTNC ARCCNGGNGA RTAYATHACN AARGTNTTYG TNGCNTTYCA RG - #CNTTYYTN        300                                                                          - MGNGGNATGG TNATGTAYAC NWSNAARGAY MGNTAYTTYT AYTTYGGNAA RY - #TNGAYGGN        360                                                                          - CARATHWSNW SNGCNTAYCC NWSNCARGAR GGNCARGTNY TNGTNGGNAT HT - #AYGGNCAR        420                                                                          - TAYCARYTNY TNGGNATHAA RWSNATHGGN TTYGARTGGA AYTAYCCNYT NG - #ARGARCCN        480                                                                          - ACNACNGARC CNCCNGTNAA YYTNACNTAY WSNGCNAAYW SNCCNGTNGG NM - #GN              534                                                                          - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE:  ZC13703                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #  18              GG                                                         - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE:  ZC13704                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #  18              CT                                                         - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC13465                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                #                25ACG GGTGA                                                  - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC13447                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                #               25 CGGC CAGAG                                                 - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE:  ZC13448                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #               25 ACCC ACGGG                                                 - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC13449                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #               25 ATGT ATGGC                                                 - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC6583                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                #21                GAGG G                                                     - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC5020                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                #21                TCCA G                                                     - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC13735                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                #             51ACAAGAG AGATTACAAG GACGATGATG ACAAGGGTGG T                    - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 66 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC14291                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - TGGTGCTGAA ATACTTGCCT CCTCCAGGGC CATACATCTT CCCACCACCC TT - #GTCATCAT         60                                                                          #           66                                                                - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC14297                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                # 44               AAGA AGGTGTAAGC TTGGACAAGA GAGA                            - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE:  ZC14279                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #              50TCGTAG TCTTCAGTGG TGCTGAAATA CTTGCCTCCT                      - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 139 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                - AGCATTGCTG CTAAAGAAGA AGGTGTAAGC TTGGACAAGA GAGATTACAA GG - #ACGATGAT         60                                                                          - GACAAGGGTG GTGGGAAGAT GTATGGCCCT GGAGGAGGCA AGTATTTCAG CA - #CCACTGAA        120                                                                          #139               TCA                                                        - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 68 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE:  ZC14346                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                - CAGTTAATCT CACATACTCA GCAAACTCAC CCGTGGGTCG CTAGGAATTC TA - #GTATTCTA         60                                                                          #          68                                                                 - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 60 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE:  ZC14218                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                - TGGCAAACTC TCAAAAATTA TAAAAATATC CAAACAGGCA GCCCTAGAAT AC - #TAGAATTC         60                                                                          - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE:  ZC14278                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                #             51ACCACTG AGCCACCAGT TAATCTCACA TACTCAGCAA A                    - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 52 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ZC13734                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                - ATCATAGAAG AGAAAAACAT TAGTTGGCAA ACTCTCAAAA ATTATAAAAA TA - #                 52                                                                          - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 154 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                - ACTAGAGGAG CCGACCACTG AGCCACCAGT TAATCTCACA TACTCAGCAA AC - #TCACCCGT         60                                                                          - GGGTCGCTAG GAATTCTAGT ATTCTAGGGC TGCCTGTTTG GATATTTTTA TA - #ATTTTTGA        120                                                                          #       154        GTTT TTCTCTTCTA TGAT                                       __________________________________________________________________________

What is claimed is:
 1. An isolated polypeptide comprising a sequence ofamino acid residues that is at least 80% identical in amino acidsequence to residues 23-178 of SEQ ID NO:2.
 2. An isolated polypeptideaccording to claim 1, wherein said polypeptide is at least 90% identicalin amino acid sequence to residues 23-178 of SEQ ID NO:2.
 3. An isolatedpolypeptide according to claim 1, wherein said polypeptide comprisesresidues 7-178 of SEQ ID NO:2.
 4. An isolated polypeptide according toclaim 1, wherein said polypeptide comprises residues 1-178 of SEQ IDNO:2.
 5. An isolated polypeptide according to claim 1, covalently linkedto a moiety selected from the group consisting of affinity tags, toxins,radionucleotides, enzymes, and fluorophores.
 6. An isolated polypeptideaccording to claim 5, wherein said affinity tag is selected from thegroup consisting of polyhistidine, FLAG, Glu-Glu, glutathione Stransferase, and an immunoglobulin heavy chain constant region.
 7. Anisolated polypeptide according to claim 6 further comprising aproteolytic cleavage site between said sequence of amino acid residuesand said affinity tag.
 8. A pharmaceutical composition comprising apolypeptide that is at least 80% identical in amino acid sequence toresidues 23-178 of SEQ ID NO:2, in combination with a pharmaceuticallyacceptable vehicle.